Dosing regimen for treatment of cognitive and motor impairments with blood plasma and blood plasma products

ABSTRACT

Methods and compositions for treating and/or preventing aging-related conditions are described. The compositions used in the methods include blood plasma and blood plasma fractions derived from blood plasma with efficacy in treating and/or preventing aging-related conditions such as cognitive disorders. The methods relate to a regimen of pulsed dosing of blood plasma or blood plasma fractions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/432,114 filed Jun. 5, 2019, now issued as U.S. Pat. No. 10,874,692,which application is a continuation of U.S. application Ser. No.15/961,618 filed Apr. 24, 2018, now issued as U.S. Pat. No. 10,357,513,which application, pursuant to 35 U.S.C. § 119 (e), claims priority tothe filing dates of: U.S. Provisional Patent Application No. 62/490,519filed Apr. 26, 2017; U.S. Provisional Patent Application No. 62/584,571filed Nov. 10, 2017; U.S. Provisional Patent Application No. 62/623,468filed Jan. 29, 2018; and U.S. Provisional Patent Application No.62/641,194 filed Mar. 9, 2018; the disclosures of which applications areherein incorporated by reference.

FIELD

This invention pertains to the prevention and treatment ofaging-associated disease. The invention relates to the use of bloodproducts, such as blood plasma fractions to treat and/or preventconditions associated with aging, such as cognitive disorders, motordisorders, and neuroinflammation using various dosing paradigms.

BACKGROUND

The following is offered as background information only and is notadmitted as prior art to the present invention.

Aging is an important risk factor for multiple human diseases includingcognitive impairment, cancer, arthritis, vision loss, osteoporosis,diabetes, cardiovascular disease, and stroke. In addition to normalsynapse loss during natural aging, synapse loss is an early pathologicalevent common to many neurodegenerative conditions and is the bestcorrelate to the neuronal and cognitive impairment associated with theseconditions. As such, aging remains the single most dominant risk factorfor dementia-related neurodegenerative diseases such as Alzheimer'sdisease (AD) (Bishop, N. A. et al., Neural mechanisms of ageing andcognitive decline. Nature 464(7288), 529-535 (2010); Heeden, T. et al.,Insights into the ageing mind: a view from cognitive neuroscience. Nat.Rev. Neurosci. 5(2), 87-96 (2004); Mattson, M. P., et al., Ageing andneuronal vulnerability. Nat. Rev. Neurosci. 7(4), 278-294 (2006)). Agingaffects all tissues and functions of the body including the centralnervous system, and neurodegeneration and a decline in functions such ascognition or motor skills, can severely impact quality of life.Treatment for cognitive decline, motor impairment, and neurodegenerativedisorders has had limited success in preventing and reversingimpairment. It is therefore important to identify new treatments formaintaining cognitive integrity by protecting against, countering, orreversing the effects of aging. Further, when new treatments aredeveloped, dosing paradigms must be investigated to optimize theefficacy of those treatments.

Although parabiosis experiments between old and young mice have shownthat cognitive function can be improved in old mice in heterochronicblood exchange with young mice, recent reports find that there is noenhancement of neurogenesis in old mice by one exchange of young blood.(Rebo, J. et al. A single heterochronic blood exchange reveals rapidinhibition of multiple tissues by old blood. Nat. Comm (2016)). Further,there is doubt that cognitive function resulting from infusions of youngplasma and neurogenesis are linked. Thus, a dosing regimen using bloodplasma or blood plasma fractions that stimulates neurogenesis andimproved cognitive function had yet to be described.

SUMMARY

The present invention is based on the production and use of bloodproducts for treating and/or preventing age-related disorders, such ascognitive impairment conditions, age-related dementia, impairment ofmotor function, neuroinflammation, and neurodegenerative disease. Thepresent invention recognizes, among other things, the need for newtherapies for the treatment and/or prevention of cognitive impairment,age-related dementia, motor impairment, neuroinflammation, andneurodegenerative disease. Derived from blood and blood plasma, thepresent compositions of the invention relate to a solution for thefailures and shortcomings of current therapies through utilization ofblood plasma fractions exhibiting efficacy in the treatment and/orprevention of cognitive impairment, age-related dementia, motorimpairment, neuroinflammation, and neurodegenerative disease.

The invention recognizes that blood plasma proteins have an averagehalf-life of 2-3 days. The invention uses a blood plasma or PlasmaFraction dosing regimen that optimizes neurogenesis, cell survival,decline in neuroinflammation, and improved cognition or motor functionin the treated subject. The dosing regimen of the invention has beenfound to trigger all of these processes (neurogenesis, cell survival,improved cognition, decreased neuroinflammation, and improved motorfunction) in subjects, and the processes have all been found to beactive even weeks after the final dose.

An embodiment of the invention includes treating a subject diagnosedwith a cognitive impairment by administering to the subject an effectiveamount of blood plasma or Plasma Fraction. Another embodiment of theinvention includes administering the effective amount of blood plasma orPlasma Fraction and subsequently monitoring the subject for improvedcognitive function. Another embodiment of the invention includestreating a subject diagnosed with a cognitive impairment byadministering to the subject an effective amount of blood plasma orPlasma Fraction wherein the blood plasma or Plasma Fraction isadministered in a manner resulting in improved cognitive function orneurogenesis.

An embodiment of the invention includes treating a subject diagnosedwith a neurodegenerative motor disorder such as, by way of example andnot limitation Parkinson's Disease, by administering to the subject aneffective amount of blood plasma or Plasma Fraction. Another embodimentof the invention includes administering the effective amount of bloodplasma or Plasma Fraction and subsequently monitoring the subject forimproved motor function. Another embodiment of the invention includestreating a subject diagnosed with a neurodegenerative motor disorder byadministering to the subject an effective amount of blood plasma orPlasma Fraction wherein the blood plasma or Plasma Fraction isadministered in a manner resulting in improved motor function orneurogenesis.

An embodiment of the invention includes treating a subject diagnosedwith neuroinflammation or a neuroinflammation-associated disorder byadministering to the subject an effective amount of blood plasma orPlasma Fraction. Another embodiment of the invention includesadministering the effective amount of blood plasma or Plasma Fractionand subsequently monitoring the subject for reduced neuroinflammation.Another embodiment of the invention includes treating a subjectdiagnosed with neuroinflammation or a neuroinflammation-associateddisorder by administering to the subject an effective amount of bloodplasma or Plasma Fraction wherein the blood plasma or Plasma Fraction isadministered in a manner resulting in reduced neuroinflammation.

Another embodiment of the invention includes administering the bloodplasma or Plasma Fraction via a dosing regimen of at least twoconsecutive days. A further embodiment of the invention includesadministering the blood plasma or Plasma Fraction via a dosing regimenof at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 consecutive days(referred to as “Pulsed Dosing,” “Pulsed Dose,” “Pulse Dosing,” “PulseDose,” or “Pulse Dosed” herein). Yet another embodiment of the inventionincludes administering the blood plasma or Plasma Fraction via a dosingregimen of at least 2 consecutive days and after the date of lastadministration. Another embodiment of the invention includesadministering the blood plasma or Plasma Fraction via a dosing regimenof 2 to 14 non-consecutive days wherein each gap between doses may bebetween 0-3 days each. Another embodiment of the invention includesmonitoring the subject for improved cognitive or motor function,decreased neuroinflammation, or improved neurogenesis at least 3 daysafter the date of last administration. Another embodiment of theinvention includes monitoring the subject for improved cognitive ormotor function, decreased neuroinflammation, or improved neurogenesisbeyond when the average half-life of the proteins in the blood plasma orPlasma Fraction has been reached.

In some instances, Pulsed Dosing in accordance with the inventionincludes administration of a first set of doses, e.g., as describedabove, followed by a period of no dosing, e.g., a “dosing-free period”,which in turn is followed by administration of another dose or set ofdoses. The duration of this “dosing-free” period, may vary, but in someembodiments, is 7 days or longer, such as 10 days or longer, including14 days or longer, wherein some instances the dosing-free period rangesfrom 15 to 365 days, such as 30 to 90 days and including 30 to 60 days.As such, embodiments of the methods include non-chronic (i.e.,non-continuous) dosing, e.g., non-chronic administration of a bloodplasma product. In some embodiments, the pattern of Pulsed Dosingfollowed by a dosing-free period is repeated for a number of times, asdesired, where in some instances this pattern is continued for 1 year orlonger, such as 2 years or longer, up to and including the life of thesubject. Another embodiment of the invention includes administering theblood plasma or Plasma Fraction via a dosing regimen of 5 consecutivedays, with a dosing-free period of 2-3 days, followed by administrationfor 2-14 consecutive days.

The current invention also recognizes that differences in proteincontent between different blood plasma fractions (e.g. fractions,effluents, Plasma Protein Fraction, Human Albumin Solution) can beresponsible for preventing and/or improving certain cognitive or motorimpairments and alleviating neurodegenerative disease. By way ofexample, and not limitation, embodiments of the current inventiondemonstrate that mere higher albumin concentration of recombinant humanalbumin or Human Albumin Solution (HAS) preparations is not the drivingforce behind improved cognition, improved motor function, reducedneuroinflammation, cell survival, or neurogenesis associated with PlasmaProtein Fraction (PPF) preparations with lower albumin concentrations.

Blood and blood plasma from young donors have exhibited improvement andreversal of the pre-existing effects of brain aging, including at themolecular, structural, functional, and cognitive levels. (Saul A.Villeda, et al. Young blood reverses age-related impairments incognitive function and synaptic plasticity in mice. Nature Medicine 20659-663 (2014)). The present invention relates to fractions andeffluents of the blood plasma, some of which have been traditionallyused to treat patient shock, and the discovery that they are effectiveas methods of treatment of aging-associated cognitive impairment,reduced motor function, and neuroinflammation orneurodegenerative-related disease.

In accordance with aspects of the invention, then, methods of treatmentof aging-associated cognitive impairment, age-related dementia, motorimpairment, neuroinflammation, and/or neurodegenerative disease usingblood product fractions of blood plasma are provided. Aspects of themethods include administering a blood plasma fraction to an individualsuffering from or at risk of developing aging-associated cognitiveimpairment, motor impairment, neuroinflammation, or neurodegenerativedisease. Additional aspects of the methods include administering a bloodplasma fraction derived from a pool of donors of a specific age range toan individual suffering from or at risk of developing aging-associatedcognitive impairment, motor impairment, neuroinflammation, orneurodegenerative disease. Further aspects of the methods includeadministration of blood plasma or Plasma Fractions using a Pulsed Dosingregimen. Also provided are reagents, devices, and kits thereof that finduse in practicing the subject methods.

In an embodiment, the blood plasma fraction may be, for example, one ofseveral blood plasma fractions obtained from a blood fractionationprocess, such as the Cohn fractionation process described below. Inanother embodiment, the blood plasma fraction may be of the type, hereinreferred to as “Plasma Fraction,” which is a solution comprised ofnormal human albumin, alpha and beta globulins, gamma globulin, andother proteins, either individually or as complexes. In anotherembodiment, the blood plasma fraction may be a type of blood plasmafraction known to those having skill in the art as a “Plasma ProteinFraction” (PPF). In another embodiment, the blood plasma fraction may bea “Human Albumin Solution” (HAS) fraction. In yet another embodiment,the blood plasma fraction may be one in which substantially all of theclotting factors are removed in order to retain the efficacy of thefraction with reduced risk of thromboses. Embodiments of the inventionmay also include administering, for example, a fraction derived from ayoung donor or pools of young donors. Another embodiment of theinvention may include the monitoring of cognitive improvement, improvedmotor function, decreased neuroinflammation, or increased neurogenesisin a subject treated with a blood plasma fraction.

An embodiment of the invention includes treating a subject diagnosedwith a cognitive impairment, neurodegenerative motor impairment, or aneuroinflammation-associated disease by administering to the subject aneffective amount of blood plasma or Plasma Fraction. Another embodimentof the invention includes administering the effective amount of bloodplasma or Plasma Fraction and subsequently monitoring the subject forimproved cognitive function, improved motor function, decreasedneuroinflammation, or increased neurogenesis. Another embodiment of theinvention includes administering the blood plasma or Plasma Fraction viaa dosing regimen of at least two consecutive days and monitoring thesubject for improved cognitive function, improved motor function,decreased neuroinflammation, or increased neurogenesis at least 2 daysafter the date of last administration. A further embodiment of theinvention includes administering the blood plasma or Plasma Fraction viaa dosing regimen of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14days and monitoring the subject for improved cognitive function,improved motor function, decreased neuroinflammation, or increasedneurogenesis at least 3 days after the date of last administration. Yetanother embodiment of the invention includes administering the bloodplasma or Plasma Fraction via a dosing regimen of a least 2 consecutivedays and after the date of last administration, monitoring for cognitiveimprovement, improved motor function, decreased neuroinflammation, orincreased neurogenesis after the average half-life of the proteins inthe blood plasma or Plasma Fraction has been reached.

An embodiment of the invention includes treating a subject diagnosedwith a cognitive impairment, impaired motor function, neuroinflammation,or a decline in neurogenesis by administering to the subject aneffective amount of blood plasma or Plasma Fraction, with the subjectfollowing an exercise regimen after the administration. Anotherembodiment of the invention includes following an exercise regimen thatis prescribed to the subject. Another embodiment of the inventionincludes the subject exercising at a higher intensity and/or greaterfrequency than the subject exercised preceding the administration.Another embodiment of the invention includes the subject exercising at asimilar intensity and/or frequency as the subject exercised precedingthe administration.

An embodiment of the invention includes treating a subject diagnosedwith a cognitive impairment, impaired motor function, neuroinflammation,or a decline in neurogenesis by administering to the subject aneffective amount of blood plasma or Plasma Fraction in a subject who isundergoing, will undergo, or has received stem cell therapy. Anotherembodiment of the invention includes administering to a subject aneffective amount of blood plasma or Plasma Fraction where the subject isundergoing, will undergo, or has received stem cell therapy, and whereinthe stem cells used in the therapy can be embryonic stem cells,non-embryonic stem cells, induced pluripotent stem cells (iPSCs), cordblood stem cells, amniotic fluid stem cells, and the like. Anotherembodiment of the invention includes treating a subject diagnosed withtraumatic spinal cord injury, stroke, retinal disease, Huntington'sdisease, Parkinson's Disease, Alzheimer's Disease, hearing loss, heartdisease, rheumatoid arthritis, or severe burns, and who is undergoing,will undergo, or has received stem cell therapy, with an effectiveamount of blood plasma or Plasma Fraction.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts distance traveled in an open field test in mice treatedwith PPF1 using Pulse Dose and 3×/week dosing regimens.

FIG. 1B depicts time spent in the center of the open field in micetreated with PPF1 using Pulse Dose and 3×/week dosing regimens.

FIG. 2 depicts the body weight over time for mice treated with PPF1using Pulse Dose and 3×/week dosing regimens.

FIG. 3 reports the number of DCX labeled cells within the granule layerof the dentate gyrus in mice treated with PPF1 using Pulse Dose or3×/week dosing regimens.

FIG. 4 reports the number of BrdU labeled cells within the granule layerof the dentate gyrus in mice treated with PPF1 using Pulse Dose or3×/week dosing regimens.

FIG. 5 reports the number of DCX labeled cells within the granule layerof the dentate gyrus in mice treated with PPF1 using Pulse Dose or3×/week dosing regimens, young human plasma (“YP”), or old human plasma(“OP”).

FIG. 6 reports the number of BrdU labeled cells within the granule layerof the dentate gyrus in mouse groups treated with PPF1 using Pulse Doseor 3×/week dosing regimens, YP, or OP.

FIG. 7 reports the latency to find the target hole per trial per day formice Pulse Dosed with PPF1 or YP.

FIG. 8 reports the number of DCX labeled cells within the granule layerof the dentate gyrus in groups of mice treated with either young humanplasma (YP), old human plasma (OP), or PPF1 using a Pulse Dosed regimen.

FIG. 9 reports the number of BrdU labeled cells within the granule layerof the dentate gyrus in groups of mice treated with either young humanplasma (YP), old human plasma (OP), or PPF1 using a Pulse Dosed regimen.

FIG. 10 reports the percent of total number of entries made into eitherthe familiar or novel arm of total entries made into each arm bytreatment group in the Y-maze test. Twelve-month-old mice were PulseDose treated with PPF1 or 5× concentrated PPF1.

FIG. 11 reports the ratio of bouts into the novel versus the familiararm of the Y-maze test. Twelve-month-old mice were Pulse Dose treatedwith PPF1 or 5× concentrated PPF1.

FIG. 12 reports the number of BrdU labeled cells per hippocampal sectionin twelve-month-old mice that were Pulse Dosed with PPF1 or 5×concentrated PPF1.

FIG. 13 reports the number of DCX labeled cells per hippocampal sectionin twelve-month-old mice that were Pulse Dosed with PPF1 or 5×concentrated PPF1.

FIG. 14 reports the number of DCX labeled cells within the granule layerof the dentate gyrus in 10.5 month-old NSG mice that were Pulse Dosedwith PPF1 or saline using one of the following regimens: (1) 5sequential days [PPF1-5d]; (2) 7 sequential days [PPF1-7d]; (3) 5sequential days with an additional 5 sequential days (“booster”) ofdosing occurring 6 weeks after the completion of the initial dosing[PPF1-5d-B]; or (4) 7 sequential days with an additional 7 sequentialdays (“booster”) of dosing occurring 6 weeks after the completion of theinitial dosing [PPF1-7d-B].

FIG. 15 reports the number of BrdU labeled cells within the granulelayer of the dentate gyrus in 10.5 month-old NSG mice that were PulseDosed with PPF1 or saline using one of the following regimens: (1) 5sequential days [PPF1-5d]; (2) 7 sequential days [PPF1-7d]; (3) 5sequential days with an additional 5 sequential days (“booster”) ofdosing occurring 6 weeks after the completion of the initial dosing[PPF1-5d-B]; or (4) 7 sequential days with an additional 7 sequentialdays (“booster”) of dosing occurring 6 weeks after the completion of theinitial dosing [PPF1-7d-B].

FIG. 16 reports the number of EdU labeled cells within the granule layerof the dentate gyrus in 10.5 month-old NSG mice that were Pulse Dosedwith PPF1 or saline using one of the following regimens: (1) 5sequential days [PPF1-5d]; (2) 7 sequential days [PPF1-7d]; (3) 5sequential days with an additional 5 sequential days (“booster”) ofdosing occurring 6 weeks after the completion of the initial dosing[PPF1-5d-B]; or (4) 7 sequential days with an additional 7 sequentialdays (“booster”) of dosing occurring 6 weeks after the completion of theinitial dosing [PPF1-7d-B].

FIG. 17 reports the number of DCX labeled cells within the granule layerof the dentate gyrus in 3 and 6-month-old NSG animals treated with PPF1or saline with or without running wheels.

FIG. 18 reports the number of Ki67 positively-labeled cells within thegranule layer of the dentate gyrus in 3 and 6-month-old NSG animalstreated with PPF1 or saline with or without running wheels.

FIG. 19 reports the number of BrdU positively-labeled cells within thegranule layer of the dentate gyrus in 3 and 6-month-old NSG animalstreated with PPF1 or saline with or without running wheels.

FIG. 20 reports the number of wheel revolutions during given timeperiods in 11-month-old NSG mice Pulse Dosed with either PPF1 or salinecontrol. Shaded areas indicating a dark cycle, and boxed region when ahot plate test was administered.

FIG. 21A shows the number of BrdU labeled cells within the granule layerof the dentate gyrus in three treatment groups of 10.5-month-old NSGmice, treated with young plasma, recombinant human albumin(“rhAlbumin”), and saline control.

FIG. 21B shows the number of DCX labeled cells in the granule layer ofthe dentate gyrus for three treatment groups of 10.5-month-old NSG mice,treated with young plasma, recombinant human albumin (“rhAlbumin”), andsaline control.

FIG. 22 reports the degree of increase in neuronal network activity indissociated mixed neuronal cells derived from mouse E16 cortex treatedwith control, PPF1, HAS1, or rhAlbumin.

FIG. 23 depicts four paradigms of administration of clarified old humanplasma (old plasma) or saline administered to 8-week-old (young) NSGmice.

FIG. 24A depicts VCAM-1 positive labeling in the hippocampus in8-week-old (young) NSG mice treated with twice weekly dosing of oldplasma, 48 hours after the last dose was administered.

FIG. 24B depicts VCAM-1 positive labeling in the hippocampus in8-week-old (young) NSG mice treated with thrice weekly dosing of oldplasma, 48 hours after the last dose was administered.

FIG. 24C depicts VCAM-1 positive labeling in the hippocampus in8-week-old (young) NSG mice treated with Pulsed Dosing of old plasma, 48hours after the last dose was administered.

FIG. 24D depicts VCAM-1 positive labeling in the hippocampus in8-week-old (young) NSG mice treated with Pulsed Dosing of old plasma, 21days after the last dose was administered.

FIG. 25A depicts the number of DCX-positive cells in the dentate gyrusin 8-week-old (young) NSG mice treated with twice weekly dosing of oldplasma, 48 hours after the last dose was administered.

FIG. 25B depicts the number of DCX-positive cells in the dentate gyrusin 8-week-old (young) NSG mice treated with thrice weekly dosing of oldplasma, 48 hours after the last dose was administered.

FIG. 25C depicts the number of DCX-positive cells in the dentate gyrusin 8-week-old (young) NSG mice treated Pulsed Dosing of old plasma, 21days after the last dose was administered.

FIG. 26 shows the Barnes Maze escape latency time course and reports thetime to reach and enter the escape hole for old plasma andsaline-treated 8-week-old (young) NSG mice. The mice were treated for 7consecutive days with old human plasma or saline and tested 4 weeksafter the last injection.

FIG. 27 depicts the average escape latency in the last three Barnes Mazetrials on day 4 of testing of 8-week-old (young) NSG mice who weretreated for 7 consecutive days with old human plasma or saline. Testingoccurred 4 weeks after the last injection.

FIG. 28 depicts the difference in escape latency between Barnes Mazetrials 1 and 3 in 8-week-old (young) NSG mice who were treated for 7consecutive days with old human plasma or saline. Testing occurred 4weeks after the last injection.

FIG. 29 reports the results of quantitative polymerase chain reaction(qPCR), quantifying mRNA levels of DCX, vesicular glutamate receptor(vglut1), synapsin 1 (syn1), beta III tubulin (tuj1), and brain-derivedneurotrophic factor (bdnf) in 8-week-old (young) NSG mice who weretreated for 7 consecutive days with old human plasma or saline.

FIG. 30 depicts the dosing paradigm for 8-week-old (young) NSG micetreated with 35 mg/kg of Kainic acid or saline, and subsequently treatedwith either PPF1 or saline daily for 5 consecutive days.

FIG. 31A reports the percent of CD68 positive area in the CA1 region ofthe hippocampus of mice treated as per the paradigm depicted in FIG. 28.

FIG. 31B reports the percent GFAP positive area in the CA1 region of thehippocampus of mice treated as per the paradigm depicted in FIG. 28.

FIG. 32 reports the number of cells stained for BrdU in the dentategyrus in 6-month-old NSG mice pulse dosed with PPF1 or saline controlfor 7 consecutive days with concurrent administration of BrdU. The firsttwo columns constitute a cohort analyzed 7 days after the last treatmentof PPF1/saline control and BrdU; the second two columns constitute acohort analyzed 14 days after the last treatment of PPF1/saline controland BrdU.

FIG. 33 depicts the increase in proliferating cells (Ki67+) in thedentate gyrus of 6-month-old NSG mice 10 days after completion of aPulse Dose regimen with PPF1.

FIG. 34 shows sections of the dentate gyrus and subventricular zone of6-month-old NSG mice 10 days after completion of a Pulse Dose regimenwith PPF1.

FIG. 35A reports the cell fate of cells in the dentate gyrus in6-month-old NSG mice treated with either PPF1 or saline control with a7-day Pulse Dosing regimen, where BrdU was administered for 5consecutive days immediately prior to the commencement of the PulseDosing regimen. The degree of NeuN+co-localization staining with BrdUindicates the degree to which neuroprogenitor cells became neurons. Thedegree of GFAP+co-localization staining with BrdU indicates the degreeto which neuroprogenitor cells became astrocytes.

FIG. 35B reports results from a similar experiment as FIG. 35A, but in12-month-old NSG mice.

FIG. 36A reports the cell fate of cells in the dentate gyrus in3-month-old NSG mice treated with either old plasma or saline controlwith a 7-day Pulse Dosing regimen, where BrdU was administered for 5consecutive days immediately prior to the commencement of the PulseDosing regimen. The degree of NeuN+co-localization staining with BrdUindicates the degree to which neuroprogenitor cells became neurons.

FIG. 36B reports results from the experiment detailed in FIG. 36A, butreports the degree of GFAP+co-localization staining with BrdU,indicating the degree to which neuroprogenitor cells became astrocytes

FIGS. 37A-37C report the number of cFos-positive cells in the (FIG. 37A)whole brain, (FIG. 37B) cortex, and (FIG. 37C) isocortex of 18-month-oldmice treated with a 7-day Pulse Dosing regimen of PPF1 or saline.

FIGS. 38A-38D report the number of cFos-positive cells in the (FIG. 38A)frontal cortex, (FIG. 38B) orbital cortex, (FIG. 38C) infralimbiccortex, and (FIG. 38D) prelimbic cortex of 18-month-old mice treatedwith a 7-day Pulse Dosing regimen of PPF1 or saline.

FIGS. 39A and 39B report the number of cFos-positive cells in the (FIG.39A) accessory olfactory nucleus and the (FIG. 39B) olfactory tubercleof 18-month-old mice treated with a 7-day Pulse Dosing regimen of PPF1or saline.

FIG. 40 depicts a Voxel statistics-based visualization of local corticalactivation in the frontal cortex (FRP), the orbital cortex (ORB), theinfralimbic cortex (ILA), the prelimbic cortex (PL), and the accessoryolfactory nucleus (AON) of 18-month-old mice treated with a 7-day PulseDosing regimen of PPF1 or saline.

FIG. 41A reports the percent CD68 immunoreactive area in the hippocampusin 22-month-old C57BL/6J wild type mice treated with a 7-day PulseDosing regimen with PPF1 or saline control.

FIG. 41B reports the percent Iba-1 immunoreactive area in thehippocampus in 22-month-old C57BL/6J wild type mice treated with a 7-dayPulse Dosing regimen with PPF1 or saline control.

FIG. 41C reports the percent GFAP immunoreactive area in the hippocampusin 22-month-old C57BL/6J wild type mice treated with a 7-day PulseDosing regimen with PPF1 or saline control.

FIG. 42A reports the percent change in BrdU staining in PPF1-treated23-month-old wild type C57BL/6J mice compared to saline control 6, 9,and 12 weeks post-dosing using a seven-consecutive day Pulsed Dosingregimen.

FIG. 42B reports the percent change in DCX staining in PPF1-treated23-month-old wild type C57BL/6J mice compared to saline control 6, 9,and 12 weeks post-dosing using a seven-consecutive day Pulsed Dosingregimen.

FIGS. 43A and 43B report the results of body weight measurements of 4 to4.5-month-old male alpha-synuclein mice (Line 61) (a model forParkinson's Disease) treated with a seven-consecutive day Pulsed Dosingregimen using PPF1 or vehicle control.

FIG. 44 reports the results of nest building in 4 to 4.5-month-old malealpha-synuclein mice (Line 61) (a model for Parkinson's Disease) treatedwith a seven-consecutive day Pulsed Dosing regimen using PPF1 or vehiclecontrol.

FIGS. 45A and 45B report the results of pasta gnawing and associatedmotor improvement, respectively, in 4 to 4.5-month-old malealpha-synuclein mice (Line 61) (a model for Parkinson's Disease) treatedwith a seven-consecutive day Pulsed Dosing regimen using PPF1 or vehiclecontrol.

FIG. 46 reports the results of a wire suspension test in 4 to4.5-month-old male alpha-synuclein mice (Line 61) (a model forParkinson's Disease) treated with a seven-consecutive day Pulsed Dosingregimen using PPF1 or vehicle control.

FIG. 47A shows different beam shapes and sizes used in five differentbeam walk trials of increasing difficulty.

FIG. 47B reports the results of five different beam walk trials in 4 to4.5-month-old male alpha-synuclein mice (Line 61) (a model forParkinson's Disease) treated with a seven-consecutive day Pulsed Dosingregimen using PPF1 or vehicle control. The beam walk trials wereperformed 72 hours after the last treatment dose.

FIG. 47C reports the results of five different beam walk trials in 4 to4.5-month-old male alpha-synuclein mice (Line 61) (a model forParkinson's Disease) treated with a seven-consecutive day Pulsed Dosingregimen using PPF1 or vehicle control. The beam walk trials wereperformed 3 weeks after the last treatment dose.

FIGS. 48A through 48F report histological results of striatal andhippocampal staining in 4 to 4.5-month-old male alpha-synuclein mice(Line 61) (a model for Parkinson's Disease) treated with aseven-consecutive day Pulsed Dosing regimen using PPF1 or vehiclecontrol. Histological markers examined include CD68, Iba-1, and NeuN.

FIG. 49 reports Barnes maze escape latency in 12-month-old NSG micetreated with a seven-consecutive day Pulsed Dosing regimen using PPF1,HAS1, or vehicle control.

DETAILED DESCRIPTION

1. Introduction

The present invention relates to the identification and discovery ofmethods and compositions for the treatment and/or prevention ofcognitive and motor impairment, including age-associated dementia ordecline in motor function and/or neurodegenerative disease. Describedherein are methods and compositions for the treatment of subjectssuffering from such disorders, which are aspects of the presentinvention. Also described herein are dosing regimens which triggerneurogenesis or decreased neuroinflammation and/or cognitive or motorimprovement in subjects suffering from cognitive or motor impairment.The methods and compositions described herein are useful in: preventingcognitive or motor impairment, age-associated dementia,neuroinflammation, and/or neurodegenerative disease; ameliorating thesymptoms of cognitive or motor impairment, age-associated dementia,neuroinflammation, and/or neurodegenerative disease; slowing progressionof aging-associated cognitive or motor impairment, age-associateddementia, neuroinflammation and/or neurodegenerative disease; and/orreversing the progression of aging-associated cognitive or motorimpairment, age-associated dementia, neuroinflammation, and/orneurodegenerative disease. An implementation of the invention includesusing blood plasma fractions as treatment, such as one or more fractionsor effluents obtained from blood fractionation processes, e.g., like theCohn fractionation process described below. An embodiment of theinvention includes using Plasma Fraction (a solution comprised of normalhuman albumin, alpha and beta globulins, gamma globulin, and otherproteins either individually or as complexes, hereinafter referred to as“Plasma Fraction”). Another embodiment of the invention includes usingPlasma Protein Fraction (PPF) as treatment. Another embodiment of theinvention includes using Human Albumin Solution (HAS) fraction astreatment. Yet another embodiment includes using effluents from bloodfractionation processes such as Effluent I or Effluent II/III describedbelow. An additional embodiment includes a blood plasma fraction fromwhich substantially all the clotting factors have been removed in orderto retain efficacy while reducing the risk of thromboses (for example,see U.S. Patent Application Nos. 62/236,710 and 63/376,529, which areincorporated by reference in their entirety herein).

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to a particular method orcomposition described, as such may, of course, vary. It is alsounderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

It is noted that the claims may be drafted to exclude any optionalelement. As such, this statement is intended to serve as antecedentbasis for use of such exclusive terminology as “solely,” “only” and thelike in connection with the recitation of claim elements or use of a“negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein have discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or the spirit of thepresent invention. Any recited method can be carried out in the order ofevents recited or in any other order which is logically possible.

2. Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one having ordinaryskill in the art to which the invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, some potentialand preferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “the peptide”includes reference to one or more peptides and equivalents thereof, e.g.polypeptides, known to those having skill in the art, and so forth.

In describing methods of the present invention, the terms “host”,“subject”, “individual” and “patient” are used interchangeably and referto any mammal in need of such treatment according to the disclosedmethods. Such mammals include, e.g., humans, ovines, bovines, equines,porcines, canines, felines, non-human primate, mice, and rats. Incertain embodiments, the subject is a non-human mammal. In someembodiments, the subject is a farm animal. In other embodiments, thesubject is a pet. In some embodiments, the subject is mammalian. Incertain instances, the subject is human. Other subjects can includedomestic pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats,horses, and the like), rodents (e.g., mice, guinea pigs, and rats, e.g.,as in animal models of disease), as well as non-human primates (e.g.,chimpanzees, and monkeys). As such, subjects of the invention, includebut are not limited to mammals, e.g., humans and other primates, such aschimpanzees and other apes and monkey species; and the like, where incertain embodiments the subject are humans. The term subject is alsomeant to include a person or organism of any age, weight or otherphysical characteristic, where the subjects may be an adult, a child, aninfant or a newborn.

By a “young” or “young individual” it is meant an individual that is ofchronological age of 40 years old or younger, e.g., 35 years old oryounger, including 30 years old or younger, e.g., 25 years old oryounger or 22 years old or younger. In some instances, the individualthat serves as the source of the young plasma-comprising blood productis one that is 10 years old or younger, e.g., 5 years old or younger,including 1-year-old or younger. In some instances, the subject is anewborn and the source of the plasma product is the umbilical cord,where the plasma product is harvested from the umbilical cord of thenewborn. As such, “young” and “young individual” may refer to a subjectthat is between the ages of 0 and 40, e.g., 0, 1, 5, 10, 15, 20, 25, 30,35, or 40 years old. In other instances, “young” and “young individual”may refer to a biological (as opposed to chronological) age such as anindividual who has not exhibited the levels of inflammatory cytokines inthe plasma exhibited in comparatively older individuals. Conversely,these “young” and “young individual” may refer to a biological (asopposed to chronological) age such as an individual who exhibits greaterlevels of anti-inflammatory cytokines in the plasma compared to levelsin comparatively older individuals. By way of example, and notlimitation, the inflammatory cytokine is Eotaxin, and the folddifference between a young subject or young individual and olderindividuals is at least 1.5-fold. Similarly, the fold difference betweenolder and younger individuals in other inflammatory cytokines may beused to refer to a biological age. (See U.S. patent application Ser. No.13/575,437 which is herein incorporated by reference). Usually, theindividual is healthy, e.g., the individual has no hematologicalmalignancy or autoimmune disease at the time of harvest.

By “an individual suffering from or at risk of suffering from anaging-associated cognitive impairment” is meant an individual that isabout more than 50% through its expected lifespan, such as more than60%, e.g., more than 70%, such as more than 75%, 80%, 85%, 90%, 95% oreven 99% through its expected lifespan. The age of the individual willdepend on the species in question. Thus, this percentage is based on thepredicted life-expectancy of the species in question. For example, inhumans, such an individual is 50 year old or older, e.g., 60 years oldor older, 70 years old or older, 80 years old or older, 90 years old orolder, and usually no older than 100 years old, such as 90 years old,i.e., between the ages of about 50 and 100, e.g., 50 . . . 55 . . . 60 .. . 65 . . . 70 . . . 75 . . . 80 . . . 85 . . . 90 . . . 95 . . . 100years old or older, or any age between 50-1000, that suffers from anaging-associated condition as further described below, e.g., cognitiveimpairment associated with the natural aging process; an individual thatis about 50 years old or older, e.g., 60 years old or older, 70 yearsold or older, 80 years old or older, 90 years old or older, and usuallyno older than 100 years old, i.e., between the ages of about 50 and 100,e.g., 50 . . . 55 . . . 60 . . . 65 . . . 70 . . . 75 . . . 80 . . . 85. . . 90 . . . 95 . . . 100 years old, that has not yet begun to showsymptoms of an aging-associated condition e.g., cognitive impairment; anindividual of any age that is suffering from a cognitive impairment dueto an aging-associated disease, as described further below, and anindividual of any age that has been diagnosed with an aging-associateddisease that is typically accompanied by cognitive impairment, where theindividual has not yet begun to show symptoms of cognitive impairment.The corresponding ages for non-human subjects are known and are intendedto apply herein.

As used herein, “treatment” refers to any of (i) the prevention of thedisease or disorder, or (ii) the reduction or elimination of symptoms ofthe disease or disorder. Treatment may be effected prophylactically(prior to the onset of disease) or therapeutically (following the onsetof the disease). The effect may be prophylactic in terms of completelyor partially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. Thus, the term “treatment”as used herein covers any treatment of an aging-related disease ordisorder in a mammal, and includes: (a) preventing the disease fromoccurring in a subject which may be predisposed to the disease but hasnot yet been diagnosed as having it; (b) inhibiting the disease, i.e.,arresting its development; or (c) relieving the disease, i.e., causingregression of the disease. Treatment may result in a variety ofdifferent physical manifestations, e.g., modulation in gene expression,rejuvenation of tissue or organs, etc. The therapeutic agent may beadministered before, during or after the onset of disease. The treatmentof ongoing disease, where the treatment stabilizes or reduces theundesirable clinical symptoms of the patient, is of particular interest.Such treatment may be performed prior to complete loss of function inthe affected tissues. The subject therapy may be administered during thesymptomatic stage of the disease, and in some cases after thesymptomatic stage of the disease.

In some embodiments, the aging-associated condition that is treated isan aging-associated impairment in cognitive ability in an individual. Bycognitive ability, or “cognition,” it is meant the mental processes thatinclude attention and concentration, learning complex tasks andconcepts, memory (acquiring, retaining, and retrieving new informationin the short and/or long term), information processing (dealing withinformation gathered by the five senses), visuospatial function (visualperception, depth perception, using mental imagery, copying drawings,constructing objects or shapes), producing and understanding language,verbal fluency (word-finding), solving problems, making decisions, andexecutive functions (planning and prioritizing). By “cognitive decline”,it is meant a progressive decrease in one or more of these abilities,e.g., a decline in memory, language, thinking, judgment, etc. By “animpairment in cognitive ability” and “cognitive impairment”, it is meanta reduction in cognitive ability relative to a healthy individual, e.g.,an age-matched healthy individual, or relative to the ability of theindividual at an earlier point in time, e.g., 2 weeks, 1 month, 2months, 3 months, 6 months, 1 year, 2 years, 5 years, or 10 years ormore previously. By “aging-associated cognitive impairment,” it is meantan impairment in cognitive ability that is typically associated withaging, including, for example, cognitive impairment associated with thenatural aging process, e.g., mild cognitive impairment (M. C. I.); andcognitive impairment associated with an aging-associated disorder, thatis, a disorder that is seen with increasing frequency with increasingsenescence, e.g., a neurodegenerative condition such as Alzheimer'sdisease, Parkinson's disease, frontotemporal dementia, Huntingtondisease, amyotrophic lateral sclerosis, multiple sclerosis, glaucoma,myotonic dystrophy, vascular dementia, and the like.

In some embodiments, the aging-associated condition that is treated isan aging-associated impairment in motor ability in an individual. Bymotor ability, it is meant the motor processes that include the abilityto perform complex muscle-and-nerve actions that produce movement suchas fine motor skills producing small or precise movements (e.g. writing,tying shoes) and gross motor skills for large movements (e.g. walking,running, kicking). By “motor decline”, it is meant a progressivedecrease in one or more of these abilities, e.g., a decline in findmovement or gross motor skills, etc. By “motor impaired” and “motorimpairment”, it is meant a reduction in motor ability/skills relative toa healthy individual, e.g., an age-matched healthy individual, orrelative to the ability of the individual at an earlier point in time,e.g., 2 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 5years, or 10 years or more previously. By “aging-associated motorimpairment,” it is meant an impairment or decline in motor ability thatis typically associated with aging, including, for example, motorimpairment associated with the natural aging process and motorimpairment or decline associated with an aging-associated disorder, thatis, a disorder that is seen with increasing frequency with increasingsenescence, e.g., a neurodegenerative condition such as Parkinson'sdisease, amyotrophic lateral sclerosis, and the like.

In some embodiments, the aging-associated condition that is treated isan aging-associated increase in neuroinflammation in an individual. By“neuroinflammation” it is meant biochemical and cellular responses ofthe nervous system to injury, infection, or neurodegenerative diseases.Such responses are directed at decreasing the triggering factors byinvolving central nervous system immunity to defend against potentialharm. Neurodegeneration occurs in the central nervous system andexhibits hallmarks of loss of neuronal structure and function.Neuroinflammatory diseases or neuroinflammatory-associated conditions ordiseases, includes by way of example and not limitation,neurodegenerative diseases such as Alzheimer's disease; Parkinson'sdisease, multiple sclerosis and the like.

Blood Products Comprising Plasma Components.

In practicing the subject methods, a blood product comprising plasmacomponents is administered to an individual in need thereof, e.g., anindividual suffering or at risk of suffering from a cognitive or motorimpairment, neuroinflammation and/or age-related dementia. As such,methods according to embodiments of the invention include administeringa blood product comprising plasma components from an individual (the“donor individual”, or “donor”) to an individual at least at risk ofsuffering or suffering from cognitive or motor impairment,neuroinflammation, neurodegeneration, and/or age-related dementia (the“recipient individual” or “recipient”). By a “blood product comprisingplasma components,” it is meant any product derived from blood thatcomprises plasma (e.g. whole blood, blood plasma, or fractions thereof).The term “plasma” is used in its conventional sense to refer to thestraw-colored/pale-yellow liquid component of blood composed of about92% water, 7% proteins such as albumin, gamma globulin, anti-hemophilicfactor, and other clotting factors, and 1% mineral salts, sugars, fats,hormones and vitamins. Non-limiting examples of plasma-comprising bloodproducts suitable for use in the subject methods include whole bloodtreated with anti-coagulant (e.g., EDTA, citrate, oxalate, heparin,etc.), blood products produced by filtering whole blood to remove whiteblood cells (“leukoreduction”), blood products consisting ofplasmapheretically-derived or apheretically-derived plasma, fresh-frozenplasma, blood products consisting essentially of purified plasma, andblood products consisting essentially of plasma fractions. In someinstances, plasma product that is employed is a non-whole blood plasmaproduct, by which is meant that the product is not whole blood, suchthat it lacks one or more components found in whole blood, such aserythrocytes, leukocytes, etc., at least to the extent that thesecomponents are present in whole blood. In some instances, the plasmaproduct is substantially, if not completely, acellular, where in suchinstances the cellular content may be 5% by volume or less, such as 1%or less, including 0.5% or less, where in some instances acellularplasma fractions are those compositions that completely lack cells,i.e., they include no cells.

Collection of Blood Products Comprising Plasma Components.

Embodiments of the methods described herein include administration ofblood products comprising plasma components which can be derived fromdonors, including human volunteers. The term, “human-derived” can referto such products. Methods of collection of plasma comprising bloodproducts from donors are well-known in the art. (See, e.g., AABBTECHNICAL MANUAL, (Mark A. Fung, et al., eds., 18th ed. 2014), hereinincorporated by reference).

In one embodiment, donations are obtained by venipuncture. In anotherembodiment, the venipuncture is only a single venipuncture. In anotherembodiment, no saline volume replacement is employed. In a preferredembodiment, the process of plasmapheresis is used to obtain the plasmacomprising blood products. Plasmapheresis can comprise the removal of aweight-adjusted volume of plasma with the return of cellular componentsto the donor. In the preferred embodiment, sodium citrate is used duringplasmapheresis in order to prevent cell clotting. The volume of plasmacollected from a donor is preferably between 690 to 880 mL after citrateadministration, and preferably coordinates with the donor's weight.

3. Plasma Fractions

During the Second World War, there arose a need for a stable plasmaexpander which could be employed in the battlefield when soldiers lostlarge amounts of blood. As a result, methods of preparing freeze-driedplasma were developed. However, use of freeze-dried plasma was difficultin combat situations since reconstitution required sterile water. As analternative, Dr. E. J. Cohn suggested that albumin could be used, andprepared a ready-to-use stable solution that could be introducedimmediately for treatment of shock. (See Johan, Current Approaches tothe Preparation of Plasma Fractions in (Biotechnology of Blood) 165(Jack Goldstein ed., 1st ed. 1991)). Dr. Cohn's procedure of purifyingplasma fractions utilized cold ethanol for its denaturing effect andemploys changes in pH and temperature to achieve separation.

An embodiment of the methods described herein includes theadministration of plasma fractions to a subject. Fractionation is theprocess by which certain protein subsets are separated from plasma.Fractionation technology is known in the art and relies on stepsdeveloped by Cohn et al. during the 1940s. (E. Cohn, Preparation andproperties of serum and plasma proteins. IV. A system for the separationinto fractions of the protein and lipoprotein components of biologicaltissues and fluids. 68 J Am Chem Soc 459 (1946), herein incorporated byreference). Several steps are involved in this process, each stepinvolving specific ethanol concentrations as well as pH, temperature,and osmolality shifts which result in selective protein precipitation.Precipitates are also separated via centrifugation or precipitation. Theoriginal “Cohn fractionation process” involved separation of proteinsthrough precipitates into five fractions, designated fraction I,fraction II+III, fraction IV-1, fraction IV-4 and fraction V. Albuminwas the originally identified endpoint (fraction V) product of thisprocess. In accordance with embodiments of the invention, each fraction(or effluent from a prior separation step) contains or potentiallycontains therapeutically-useful protein fractions. (See Thierry Burnouf,Modern Plasma Fractionation, 21(2) Transfusion Medicine Reviews 101(2007); Adil Denizli, Plasma fractionation: conventional andchromatographic methods for albumin purification, 4 J. Biol. & Chem.315, (2011); and T. Brodniewicz-Proba, Human Plasma Fractionation andthe Impact of New Technologies on the Use and Quality of Plasma-derivedProducts, 5 Blood Reviews 245 (1991), and U.S. Pat. Nos. 3,869,431,5,110,907, 5,219,995, 7,531,513, and 8,772,461 which are hereinincorporated by reference). Adjustment of the above experimentalparameters can be made in order to obtain specific protein fractions.

More recently, fractionation has reached further complexity, and assuch, comprises additional embodiments of the invention. This recentincrease in complexity has occurred through: the introduction ofchromatography resulting in isolation of new proteins from existingfractions like cryoprecipitate, cryo-poor plasma, and Cohn fractions;increasing IgG recovery by integrating chromatography and the ethanolfractionation process; and viral reduction/inactivation/removal. (Id.)In order to capture proteins at physiological pH and ionic strength,anion-exchange chromatography can be utilized. This preserves functionalactivity of proteins and/or protein fractions. Heparin and monoclonalantibodies are also used in affinity chromatography. One of ordinaryskill in the art would recognize that the parameters described above maybe adjusted to obtain specifically-desired plasma protein-containingfractions.

In an embodiment of the invention, blood plasma is fractionated in anindustrial setting. Frozen plasma is thawed at 1° C. to 4° C. Continuousrefrigerated centrifugation is applied to the thawed plasma andcryoprecipitate isolated. Recovered cryoprecipitate is frozen at −30° C.or lower and stored. The cryoprecipitate-poor (“cryo-poor”) plasma isimmediately processed for capture (via, for example, primarychromatography) of labile coagulation factors such as factor IX complexand its components as well as protease inhibitors such as antithrombinand C1 esterase inhibitor. Serial centrifugation and precipitateisolation can be applied in subsequent steps. Such techniques are knownto one of ordinary skill in the art and are described, for example, inU.S. Pat. Nos. 4,624,780, 5,219,995, 5,288,853, and U.S. patentapplication nos. 20140343255 and 20150343025, which disclosures areincorporated by reference in their entirety herein.

In an embodiment of the invention, the plasma fraction may comprise aplasma fraction containing a substantial concentration of albumin. Inanother embodiment of the invention, the plasma fraction may comprise aplasma fraction containing a substantial concentration of IgG orintravenous immune globulin (IGIV) (e.g. Gamunex-C®). In anotherembodiment of the invention the plasma fraction may comprise an IGIVplasma fraction, such as Gamunex-C® which has been substantiallydepleted of immune globulin (IgG) by methods well-known by one ofordinary skill in the art, such as for example, Protein A-mediateddepletion. (See Keshishian, H., et al., Multiplexed, QuantitativeWorkflow for Sensitive Biomarker Discovery in Plasma Yields NovelCandidates for Early Myocardial Injury, Molecular & Cellular Proteomics,14 at 2375-93 (2015)). In an additional embodiment, the blood plasmafraction may be one in which substantially all the clotting factors areremoved in order to retain the efficacy of the fraction with reducedrisk of thromboses. For example, the plasma fraction may be a plasmafraction as described in U.S. Patent No. 62/376,529 filed on Aug. 18,2016; the disclosure of which is incorporated by reference in itsentirety herein.

4. Albumin Products

To those having ordinary skill in the art, there are two generalcategories of Albumin Plasma Products (“APP”): plasma protein fraction(“PPF”) and human albumin solution (“HAS”). PPF is derived from aprocess with a higher yield than HAS but has a lower minimum albuminpurity than HAS (>83% for PPF and >95% for HAS). (Production of humanalbumin solution: a continually developing colloid, P. Matejtschuk etal., British J. of Anaesthesia 85(6): 887-95, at 888 (2000)). In someinstances, PPF has albumin purity of between 83% and 95% oralternatively 83% and 96%. The albumin purity can be determined byelectrophoresis or other quantifying assays such as, for example, bymass spectrometry. Additionally, some have noted that PPF has adisadvantage because of the presence of protein “contaminants” such asPKA. Id. As a consequence, PPF preparations have lost popularity asAlbumin Plasma Products, and have even been delisted from certaincountries' Pharmacopoeias. Id. Contrary to these concerns, the inventionmakes beneficial use of these “contaminants.” Besides α, β, and γglobulins, as well as the aforementioned PKA, the methods of theinvention utilize additional proteins or other factors within the“contaminants” that promote processes such as neurogenesis, neuronalcell survival, improved cognition or motor function and decreasedneuroinflammation.

Those of skill in the art will recognize that there are, or have been,several commercial sources of PPF (the “Commercial PPF Preparations.”)These include Plasma-Plex™ PPF (Armour Pharmaceutical Co., Tarrytown,N.Y.), Plasmanate™ PPF (Grifols, Clayton, N.C.), Plasmatein™ (AlphaTherapeutics, Los Angeles, Calif.), and Protenate™ PPF (Baxter Labs,Inc. Deerfield, Ill.).

Those of skill in the art will also recognize that there are, or havebeen, several commercial sources of HAS (the “Commercial HASPreparations.”) These include Albuminar™ (CSL Behring), AlbuRx™ (CSLBehring), Albutein™ (Grifols, Clayton, N.C.), Buminate™ (Baxatla, Inc.,Bannockburn, Ill.), Flexbumin™ (Baxatla, Inc., Bannockburn, Ill.), andPlasbumin™ (Grifols, Clayton, N.C.).

a. Plasma Protein Fraction (Human) (PPF)

According to the United States Food and Drug Administration (“FDA”),“Plasma Protein Fraction (Human),” or PPF, is the proper name of theproduct defined as “a sterile solution of protein composed of albuminand globulin, derived from human plasma.” (Code of Federal Regulations“CFR” 21 CFR 640.90 which is herein incorporated by reference). PPF'ssource material is plasma recovered from Whole Blood prepared asprescribed in 21 CFR 640.1-640.5 (incorporated by reference herein), orSource Plasma prepared as prescribed in 21 CFR 640.60-640.76(incorporated by reference herein).

PPF is tested to determine it meets the following standards as per 21CFR 640.92 (incorporated by reference herein):

(a) The final product shall be a 5.0+/−0.30 percent solution of protein;and

(b) The total protein in the final product shall consist of at least 83percent albumin, and no more than 17 percent globulins. No more than 1percent of the total protein shall be gamma globulin. The proteincomposition is determined by a method that has been approved for eachmanufacturer by the Director, Center for Biologics Evaluation andResearch, Food and Drug Administration.

As used herein, “Plasma Protein Fraction” or “PPF” refers to a sterilesolution of protein composed of albumin and globulin, derived from humanplasma, with an albumin content of at least 83% with no more than 17%globulins (including α1, α2, β, and γ globulins) and other plasmaproteins, and no more than 1% gamma globulin as determined byelectrophoresis. (Hink, J H, Jr., et al., Preparation and Properties ofa Heat-Treated Human Plasma Protein Fraction, VOX SANGUINIS 2(174)(1957)). PPF can also refer to a solid form, which when suspended insolvent, has similar composition. The total globulin fraction can bedetermined through subtracting the albumin from the total protein.(Busher, J., Serum Albumin and Globulin, CLINICAL METHODS: THE HISTORY,PHYSICAL, AND LABORATORY EXAMINATIONS, Chapter 10, Walker H K, Hall W D,Hurst J D, eds. (1990)).

b. Albumin (Human) (HAS)

According to the FDA, “Albumin (Human)” (also referred to herein as“HAS”) is the proper name of the product defined as “sterile solution ofthe albumin derived from human plasma.” (Code of Federal Regulations“CFR” 21 CFR 640.80 which is herein incorporated by reference.) Thesource material for Albumin (Human) is plasma recovered from Whole Bloodprepared as prescribed in 21 CFR 640.1-640.5 (incorporated by referenceherein), or Source Plasma prepared as prescribed in 21 CFR 640.60-640.76(incorporated by reference herein). Other requirements for Albumin(Human) are listed in 21 CFR 640.80-640.84 (incorporated by referenceherein).

Albumin (Human) is tested to determine if it meets the followingstandards as per 21 CFR 640.82:

(a) Protein concentration. Final product shall conform to one of thefollowing concentrations: 4.0+/−0.25 percent; 5.0+/−0.30 percent;20.0+/−1.2 percent; and 25.0+/−1.5 percent solution of protein.

(b) Protein composition. At least 96 percent of the total protein in thefinal product shall be albumin, as determined by a method that has beenapproved for each manufacturer by the Director, Center for BiologicsEvaluation and Research, Food and Drug Administration.

As used herein, “Albumin (Human)” or “HAS” refers to a to a sterilesolution of protein composed of albumin and globulin, derived from humanplasma, with an albumin content of at least 95%, with no more than 5%globulins (including α1, α2, β, and γ globulins) and other plasmaproteins. HAS can also refer to a solid form, which when suspended insolvent, has similar composition. The total globulin fraction can bedetermined through subtracting the albumin from the total protein.

As can be recognized by one having ordinary skill in the art, PPF andHAS fractions can also be freeze-dried or in other solid form. Suchpreparations, with appropriate additives, can be used to make tablets,powders, granules, or capsules, for example. The solid form can beformulated into preparations for injection by dissolving, suspending oremulsifying them in an aqueous or non-aqueous solvent, such as vegetableor other similar oils, synthetic aliphatic acid glycerides, esters ofhigher aliphatic acids or propylene glycol; and if desired, withconventional additives such as solubilizers, isotonic agents, suspendingagents, emulsifying agents, stabilizers and preservatives.

5. Clotting Factor-Reduced Fractions

Another embodiment of the invention uses a blood plasma fraction fromwhich substantially all of the clotting factors are removed in order toretain the efficacy of the fraction with reduced risk of thromboses.Conveniently, the blood product can be derived from a young donor orpool of young donors and can be rendered devoid of IgM in order toprovide a young blood product that is ABO compatible. Currently, plasmathat is transfused is matched for ABO blood type, as the presence ofnaturally occurring antibodies to the A and B antigens can result intransfusion reactions. IgM appears to be responsible for transfusionreactions when patients are given plasma that is not ABO matched.Removal of IgM from blood products or fractions helps eliminatetransfusion reactions in subjects who are administered the bloodproducts and blood plasma fractions of the invention.

Accordingly, in one embodiment, the invention is directed to a method oftreating or preventing an aging-related condition such as cognitive ormotor impairment, neuroinflammation or neurodegeneration in a subject.The method comprises: administering to the subject a blood product orblood fraction derived from whole-blood from an individual or pool ofindividuals, wherein the blood product or blood fraction issubstantially devoid of (a) at least one clotting factor and/or (b) IgM.In some embodiments, the individual(s) from whom the blood product orblood fraction is derived are young individuals. In some embodiments,the blood product is substantially devoid of at least one clottingfactor and IgM. In certain embodiments, the blood product issubstantially devoid of fibrinogen (Factor I). In additionalembodiments, the blood product substantially lacks erythrocytes and/orleukocytes. In further embodiments, the blood product is substantiallyacellular. In other embodiments, the blood product is derived fromplasma. Such embodiments of the invention are further supported by U.S.Patent Application No. 62/376,529 filed on Aug. 18, 2016, which isincorporated by reference in its entirety herein.

6. Protein-Enriched Plasma Protein Products Treatment

Additional embodiments of the invention use plasma fractions withreduced albumin concentration compared to PPF, but with increasedamounts of globulins and other plasma proteins (what have been referredto by some as “contaminants”). The embodiments, as with PPF, HAS,Effluent I, and Effluent II/III are all effectively devoid of clottingfactors. Such plasma fractions are hereinafter referred to as“protein-enriched plasma protein products”. For example, an embodimentof the invention may use a protein-enriched plasma protein productcomprised of 82% albumin and 18% α, β, and γ globulins and other plasmaproteins. Another embodiment of the invention may use a protein-enrichedplasma protein product comprised of 81% albumin and 19% of α, β, and γglobulins and/or other plasma proteins. Another embodiment of theinvention may use a protein-enriched plasma protein product comprised of80% albumin and 20% of α, β, and γ globulins and/or other plasmaproteins. Additional embodiments of the invention may useprotein-enriched plasma protein products comprised of 70-79% albumin anda corresponding 21-30% of α, β, and γ globulins and other plasmaproteins. Additional embodiments of the invention may useprotein-enriched plasma protein products comprised of 60-69% albumin anda corresponding 31-40% of α, β, and γ globulins and other plasmaproteins. Additional embodiments of the invention may useprotein-enriched plasma protein products comprised of 50-59% albumin anda corresponding 41-50% of α, β, and γ globulins and other plasmaproteins. Additional embodiments of the invention may useprotein-enriched plasma protein products comprised of 40-49% albumin anda corresponding 51-60% of α, β, and γ globulins and other plasmaproteins. Additional embodiments of the invention may useprotein-enriched plasma protein products comprised of 30-39% albumin anda corresponding 61-70% of α, β, and γ globulins and other plasmaproteins. Additional embodiments of the invention may useprotein-enriched plasma protein products comprised of 20-29% albumin anda corresponding 71-80% of α, β, and γ globulins and other plasmaproteins. Additional embodiments of the invention may useprotein-enriched plasma protein products comprised of 10-19% albumin anda corresponding 81-90% of α, β, and γ globulins and other plasmaproteins. Additional embodiments of the invention may useprotein-enriched plasma protein products comprised of 1-9% albumin and acorresponding 91-99% of α, β, and γ globulins and other plasma proteins.A further embodiment of the invention may use protein-enriched plasmaprotein products comprised of 0% albumin and 100% of α, β, and γglobulins and other plasma proteins

Embodiments of the invention described above may also have total gammaglobulin concentrations of 1-5%.

The specific concentrations of proteins in a plasma fraction may bedetermined using techniques well-known to a person having ordinary skillin the relevant art. By way of example, and not limitation, suchtechniques include electrophoresis, mass spectrometry, ELISA analysis,and Western blot analysis.

7. Preparation of Plasma Fractions

Methods of preparing PPF and other plasma fractions are well-known tothose having ordinary skill in the art. An embodiment of the inventionallows for blood used in the preparation of human plasma proteinfraction to be collected in flasks with citrate or anticoagulant citratedextrose solution for inhibition of coagulation, with further separationof Fractions I, II+III, IV, and PPF as per the method disclosed in Hinket al. (See Hink, J H, Jr., et al., Preparation and Properties of aHeat-Treated Human Plasma Protein Fraction, VOX SANGUINIS 2(174) (1957),herein incorporated by reference.) According to this method, the mixturecan be collected to 2-8° C. The plasma can then subsequently beseparated by centrifugation at 7° C., removed, and stored at −20° C. Theplasma can then be thawed at 37° C. and fractionated, preferably withineight hours after removal from −20° C. storage.

Plasma can be separated from Fraction I using 8% ethanol at pH 7.2 and atemperature at −2 to −2.5° C. with protein concentration of 5.1 to 5.6percent. Cold 53.3 percent ethanol (176 mL/L of plasma) with acetatebuffer (200 mL 4M sodium acetate, 230 mL glacial acetic acid quantumsatis to 1 L with H₂O) can be added using jets at a rate, for example,of 450 mL/minute during the lowering the plasma temperature to −2° C.Fraction I can be separated and removed from the effluent (Effluent I)through ultracentrifugation. Fibrinogen can be obtained from Fraction Ias per methods well-known to those having ordinary skill in the art.

Fraction II+III can be separated from Effluent I through adjustment ofthe effluent to 21 percent ethanol at pH 6.8, temperature at −6° C.,with protein concentration of 4.3 percent. Cold 95 percent ethanol (176mL/L of Effluent I) with 10 M acetic acid used for pH adjustment can beadded using jets at a rate, for example, of 500 mL/minute during thelowering of the temperature of Effluent I to −6° C. The resultingprecipitate (Fraction II+III) can be removed by centrifugation at −6° C.Gamma globulin can be obtained from Fraction II+III using methodswell-known to those having ordinary skill in the art.

Fraction IV-1 can be separated from Effluent II+III (“Effluent II/III”)through adjustment of the effluent to 19 percent ethanol at pH 5.2,temperature at −6° C., and protein concentration of 3 percent. H₂O and10 M acetic acid used for pH adjustment can be added using jets whilemaintaining Effluent II/III at −6° C. for 6 hours. Precipitated FractionVI-1 can be settled at −6° C. for 6 hours and subsequently separatedfrom the effluent by centrifugation at the same temperature. Stableplasma protein fraction can be recovered from Effluent IV-1 throughadjustment of the ethanol concentration to 30 percent at pH 4.65,temperature −7° C. and protein concentration of 2.5 percent. This can beaccomplished by adjusting the pH of Effluent IV-1 with cold acid-alcohol(two parts 2 M acetic acid and one-part 95 percent ethanol). Whilemaintaining a temperature of −7° C., to every liter of adjusted EffluentIV-1 170 mL cold ethanol (95%) is added. Proteins that precipitate canbe allowed to settle for 36 hours and subsequently removed bycentrifugation at −7° C.

The recovered proteins (stable plasma protein fraction) can be dried(e.g. by freeze drying) to remove alcohol and H20. The resulting driedpowder can be dissolved in sterile distilled water, for example using 15liters of water/kg of powder, with the solution adjusted to pH 7.0 with1 M NaOH. A final concentration of 5 percent protein can be achieved byadding sterile distilled water containing sodium acetyl tryptophanate,sodium caprylate, and NaCl, adjusting to final concentrations of 0.004 Macetyl tryptophanate, 0.004 M caprylate, and 0.112 M sodium. Finally,the solution can be filtered at 10° C. to obtain a clear solution andsubsequently heat-treated for inactivation of pathogens at 60° C. for atleast 10 hours.

One having ordinary skill in the art would recognize that each of thedifferent fractions and effluents described above could be used with themethods of the invention to treat disease. For example, and not by wayof limitation, Effluents I or Effluent II/III may be utilized to treatsuch diseases as cognitive, motor, and neurodegenerative disorders andare embodiments of the invention.

The preceding methods of preparing plasma fractions and plasma proteinfraction (PPF) are only exemplary and involves merely embodiments of theinvention. One having ordinary skill in the art would recognize thatthese methods can vary. For example, pH, temperature, and ethanolconcentration, among other things can be adjusted to produce differentvariations of plasma fractions and plasma protein fraction in thedifferent embodiments and methods of the invention. In another example,additional embodiments of the invention contemplate the use ofnanofiltration for the removal/inactivation of pathogens from plasmafractions and plasma protein fraction.

An additional embodiment of the invention contemplates methods andcomposition using and/or comprising additional plasma fractions. Forexample, the invention, among other things, demonstrates that specificconcentrations of albumin are not critical for improving cognitive ormotor activity. Hence, fractions with reduced albumin concentration,such as those fractions having below 83% albumin, are contemplated bythe invention.

8. Treatment

Aspects of the methods of the inventions described herein includetreatment of a subject with a plasma comprising blood product, such as ablood plasma fraction, e.g., as described above. An embodiment includestreatment of a human subject with a plasma comprising blood product. Oneof skill in the art would recognize that methods of treatment ofsubjects with plasma comprising blood products are recognized in theart. By way of example, and not limitation, one embodiment of themethods of the inventions described herein is comprised of administeringfresh frozen plasma to a subject for treatment and/or prevention ofcognitive or motor impairment, neuroinflammation, neurodegeneration,and/or age-related dementia. In one embodiment, the plasma comprisingblood product is administered immediately, e.g., within about 12-48hours of collection from a donor, to the individual suffering or at riskfrom a cognitive or motor impairment, neuroinflammation,neurodegeneration, and/or age-related dementia. In such instances, theproduct may be stored under refrigeration, e.g., 0-10° C. In anotherembodiment, fresh frozen plasma is one that has been stored frozen(cryopreserved) at −18° C. or colder. Prior to administration, the freshfrozen plasma is thawed and once thawed, administered to a subject 60-75minutes after the thawing process has begun. Each subject preferablyreceives a single unit of fresh frozen plasma (200-250 mL), the freshfrozen plasma preferably derived from donors of a pre-determined agerange. In one embodiment of the invention, the fresh frozen plasma isdonated by (derived from) young individuals. In another embodiment ofthe invention, the fresh frozen plasma is donated by (derived from)donors of the same gender. In another embodiment of the invention, thefresh frozen plasma is donated by (derived from) donors of the age rangebetween 18-22 years old.

In an embodiment of the invention, the plasma comprising blood productsare screened after donation by blood type. In another embodiment of theinvention, the plasma comprising blood products are screened forinfectious disease agents such as HIV I & II, HBV, HCV, HTLV I & II,anti-HBc per the requirements of 21 CFR 640.33 and recommendationscontained in FDA guidance documents.

In yet another embodiment of the invention, the subject is treated witha Plasma Fraction. In an embodiment of the invention, the plasmafraction is PPF or HAS. In a further embodiment of the invention, theplasma fraction is one of the Commercial PPF Preparations of theCommercial HAS Preparations. In another embodiment of the invention theplasma fraction is a PPF or HAS derived from a pool of individuals of aspecific age range, such as young individuals, or is a modified PPF orHAS fraction which has been subjected to additional fractionation orprocessing (e.g. PPF or HAS with one or more specific proteins partiallyor substantially removed). In another embodiment of the invention, theplasma fraction is an IGIV plasma fraction which has been substantiallydepleted of immune globulin (IgG). A blood fraction which is“substantially depleted” or which has specific proteins “substantiallyremoved,” such as IgG, refers to a blood fraction containing less thanabout 50% of the amount that occurs in the reference product or wholeblood plasma, such as less than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 5%,4%, 3%, 2%, 1%, 0.5%, 0.25%, 0.1%, undetectable levels, or any integerbetween these values, as measured using standard assays well known inthe art.

9. Administration

Aspects of the methods of the inventions described herein includetreatment of a subject with a plasma comprising blood product, such as ablood plasma or Plasma Fraction, e.g., as described above. An embodimentincludes treatment of a human subject with a plasma comprising bloodproduct. One of skill in the art would recognize that methods oftreatment of subjects with plasma comprising blood products arerecognized in the art. By way of example, and not limitation, oneembodiment of the methods of the inventions described herein iscomprised of administering fresh frozen plasma to a subject fortreatment and/or prevention of cognitive or motor impairment,neuroinflammation, neurodegeneration, and/or age-related dementia. Inone embodiment, the plasma comprising blood product is administeredimmediately, e.g., within about 12-48 hours of collection from a donor,to the individual suffering or at risk from a cognitive or motorimpairment, neuroinflammation, neurodegeneration, and/or age-relateddementia. In such instances, the product may be stored underrefrigeration, e.g., 0-10° C. In another embodiment, fresh frozen plasmais one that has been stored frozen (cryopreserved) at −18° C. or colder.Prior to administration, the fresh frozen plasma is thawed and oncethawed, administered to a subject 60-75 minutes after the thawingprocess has begun. Each subject preferably receives a single unit offresh frozen plasma (200-250 mL), the fresh frozen plasma preferablyderived from donors of a pre-determined age range. In one embodiment ofthe invention, the fresh frozen plasma is donated by (derived from)young individuals. In another embodiment of the invention, the freshfrozen plasma is donated by (derived from) donors of the same gender. Inanother embodiment of the invention, the fresh frozen plasma is donatedby (derived from) donors of the age range between 18-22 years old.

In an embodiment of the invention, the plasma comprising blood productsare screened after donation by blood type. In another embodiment of theinvention, the plasma comprising blood products are screened forinfectious disease agents such as HIV I & II, HBV, HCV, HTLV I & II,anti-HBc per the requirements of 21 CFR 640.33 and recommendationscontained in FDA guidance documents.

In yet another embodiment of the invention, the subject is treated witha Plasma Fraction. In an embodiment of the invention, the plasmafraction is PPF or HAS. In a further embodiment of the invention, theplasma fraction is one of the Commercial PPF Preparations of theCommercial HAS Preparations. In another embodiment of the invention theplasma fraction is a PPF or HAS derived from a pool of individuals of aspecific age range, such as young individuals, or is a modified PPF orHAS fraction which has been subjected to additional fractionation orprocessing (e.g. PPF or HAS with one or more specific proteins partiallyor substantially removed). In another embodiment of the invention, theplasma fraction is an IGIV plasma fraction which has been substantiallydepleted of immune globulin (IgG). A blood fraction which is“substantially depleted” or which has specific proteins “substantiallyremoved,” such as IgG, refers to a blood fraction containing less thanabout 50% of the amount that occurs in the reference product or wholeblood plasma, such as less than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 5%,4%, 3%, 2%, 1%, 0.5%, 0.25%, 0.1%, undetectable levels, or any integerbetween these values, as measured using standard assays well known inthe art.

An embodiment of the invention includes treating a subject diagnosedwith a cognitive or motor impairment, neurodegeneration, orneuroinflammation by administering to the subject an effective amount ofblood plasma or Plasma Fraction. Another embodiment of the inventionincludes administering the effective amount of blood plasma or PlasmaFraction and subsequently monitoring the subject for improved cognitiveor motor function, or a reduction in neuroinflammation or increase inneurogenesis. Another embodiment of the invention includes treating asubject diagnosed with a cognitive or motor impairment,neurodegeneration, or neuroinflammation by administering to the subjectan effective amount of blood plasma or Plasma Fraction wherein the bloodplasma or Plasma Fraction is administered in a manner resulting inimproved cognitive or motor function, decreased neuroinflammation, orimproved neurogenesis after the mean or median half-life of the bloodplasma proteins or Plasma Fraction proteins been reached, relative tothe most recent administered dose (referred to as “Pulsed Dosing” or“Pulse Dosed” herein). Another embodiment of the invention includesadministering the blood plasma or Plasma Fraction via a dosing regimenof at least two consecutive days and monitoring the subject for improvedcognitive or motor function, decreased neuroinflammation or improvedneurogenesis at least 3 days after the date of last administration. Afurther embodiment of the invention includes administering the bloodplasma or Plasma Fraction via a dosing regimen of at least 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, or 14 consecutive days and monitoring thesubject for improved cognitive or motor function, decreasedneuroinflammation, or increased neurogenesis at least 3 days after thedate of last administration. Yet another embodiment of the inventionincludes administering the blood plasma or Plasma Fraction via a dosingregimen of at least 2 consecutive days and after the date of lastadministration, monitoring for cognitive or motor function improvement,decreased neuroinflammation, or increased neurogenesis beyond when theaverage half-life of the proteins in the blood plasma or Plasma Fractionhas been reached. Another embodiment of the invention includesadministering the blood plasma or Plasma Fraction via a dosing regimenof 2 to 14 non-consecutive days wherein each gap between doses may bebetween 0-3 days each.

In some instances, Pulsed Dosing in accordance with the inventionincludes administration of a first set of doses, e.g., as describedabove, followed by a period of no dosing, e.g., a “dosing-free period”,which in turn is followed by administration of another dose or set ofdoses. The duration of this “dosing-free” period, may vary, but in someembodiments, is 7 days or longer, such as 10 days or longer, including14 days or longer, wherein some instances the dosing-free period rangesfrom 15 to 365 days, such as 30 to 90 days and including 30 to 60 days.As such, embodiments of the methods include non-chronic (i.e.,non-continuous) dosing, e.g., non-chronic administration of a bloodplasma product. In some embodiments, the pattern of Pulsed Dosingfollowed by a dosing-free period is repeated for a number of times, asdesired, where in some instances this pattern is continued for 1 year orlonger, such as 2 years or longer, up to and including the life of thesubject. Another embodiment of the invention includes administering theblood plasma or Plasma Fraction via a dosing regimen of 5 consecutivedays, with a dosing-free period of 2-3 days, followed by administrationfor 2-14 consecutive days.

Biochemically, by an “effective amount” or “effective dose” of activeagent is meant an amount of active agent that will inhibit, antagonize,decrease, reduce, or suppress by about 20% or more, e.g., by 30% ormore, by 40% or more, or by 50% or more, in some instances by 60% ormore, by 70% or more, by 80% or more, or by 90% or more, in some casesby about 100%, i.e., to negligible amounts, and in some instances,reverse the progression of the cognitive or impairment,neuroinflammation, neurodegeneration, or age-associated dementia.

10. Plasma Protein Fraction

In practicing methods of the invention, a plasma fraction isadministered to the subject. In an embodiment, the plasma fraction isplasma protein fraction (PPF). In additional embodiments, the PPF isselected from the Commercial PPF Preparations.

In another embodiment, the PPF is comprised of 88% normal human albumin,12% alpha and beta globulins and not more than 1% gamma globulin asdetermined by electrophoresis. Further embodiments of this embodimentused in practicing methods of the invention include, for example, theembodiment as a 5% solution of PPF buffered with sodium carbonate andstabilized with 0.004 M sodium caprylate and 0.004 M acetyltryptophan.Additional formulations, including those modifying the percentage of PPF(e.g. about 1% to about 10%, about 10% to about 20%, about 20% to 25%,about 25% to 30%) in solution as well as the concentrations of solventand stabilizers may be utilized in practicing methods of the invention.

11. Plasma Fractions of Specific Donor Age

Additional embodiments of the invention include administering a plasmaprotein fraction derived from the plasma of individuals of certain ageranges. An embodiment includes administering PPF or HAS which have beenderived from the plasma of young individuals. In another embodiment ofthe invention the young individuals are of a single specific age or aspecific age range. In yet another embodiment, the average age of thedonors is less than that of the subject or less than the average age ofthe subjects being treated.

Certain embodiments of the invention include pooling blood or bloodplasma from individuals of specific age ranges and fractionating theblood plasma as described above to attain a plasma protein fractionproduct such as PPF or HAS. In an alternate embodiment of the invention,the plasma protein fraction or specific plasma protein fraction isattained from specific individuals fitting a specified age range.

12. Indications

The subject methods and plasma-comprising blood products and fractionsfind use in treating, including preventing, aging-associated conditions,such as impairments in the cognitive or motor ability of individuals,e.g., cognitive disorders, including (but not limited to) age-associateddementia, immunological conditions, cancer, and physical and functionaldecline; and motor disorders such as (but not limited to) Parkinson'sdisease. Individuals suffering from or at risk of developing anaging-associated cognitive or motor impairment, neuroinflammation,and/or neurodegeneration that will benefit from treatment with thesubject plasma-comprising blood product, e.g., by the methods disclosedherein, include individuals that are about 50 years old or older, e.g.,60 years old or older, 70 years old or older, 80 years old or older, 90years old or older, and 100 years old or older, i.e., between the age ofabout 50 and 100, e.g., 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or about100 years old, and are suffering from cognitive or motor impairment,neuroinflammation, and/or neurodegeneration associated with naturalaging process, e.g., mild cognitive impairment (M. C. I.); andindividuals that are about 50 years old or older, e.g., 60 years old orolder, 70 years old or older, 80 years old or older, 90 years old orolder, and usually no older than 100 years old, i.e., between the agesof about 50 and 90, e.g., 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 orabout 100 years old, that have not yet begun to show symptoms ofcognitive or motor impairment, neuroinflammation and/orneurodegeneration. Examples of cognitive and motor, neuroinflammatory,and/or neurodegenerative impairments that are due to natural aginginclude the following:

a. Mild cognitive impairment (M. C. I.). Mild cognitive impairment is amodest disruption of cognition that manifests as problems with memory orother mental functions such as planning, following instructions, ormaking decisions that have worsened over time while overall mentalfunction and daily activities are not impaired. Thus, althoughsignificant neuronal death does not typically occur, neurons in theaging brain are vulnerable to sub-lethal age-related alterations instructure, synaptic integrity, and molecular processing at the synapse,all of which impair cognitive function.

Individuals suffering from or at risk of developing an aging-associatedcognitive impairment that will benefit from treatment with the subjectplasma-comprising blood product or fraction, e.g., by the methodsdisclosed herein, also include individuals of any age that are sufferingfrom a cognitive impairment due to an aging-associated disorder; andindividuals of any age that have been diagnosed with an aging-associateddisorder that is typically accompanied by cognitive impairment, wherethe individual has not yet begun to present with symptoms of cognitiveimpairment. Examples of such aging-associated disorders include thefollowing:

b. Alzheimer's disease. Alzheimer's disease is a progressive, inexorableloss of cognitive function associated with an excessive number of senileplaques in the cerebral cortex and subcortical gray matter, which alsocontains b-amyloid and neurofibrillary tangles consisting of tauprotein. The common form affects persons>60 yr old, and its incidenceincreases as age advances. It accounts for more than 65% of thedementias in the elderly.

The cause of Alzheimer's disease is not known. The disease runs infamilies in about 15 to 20% of cases. The remaining, so-called sporadiccases have some genetic determinants. The disease has an autosomaldominant genetic pattern in most early-onset and some late-onset casesbut a variable late-life penetrance. Environmental factors are the focusof active investigation.

In the course of the disease, synapses, and ultimately neurons are lostwithin the cerebral cortex, hippocampus, and subcortical structures(including selective cell loss in the nucleus basalis of Meynert), locuscoeruleus, and nucleus raphae dorsalis. Cerebral glucose use andperfusion is reduced in some areas of the brain (parietal lobe andtemporal cortices in early-stage disease, prefrontal cortex inlate-stage disease). Neuritic or senile plaques (composed of neurites,astrocytes, and glial cells around an amyloid core) and neurofibrillarytangles (composed of paired helical filaments) play a role in thepathogenesis of Alzheimer's disease. Senile plaques and neurofibrillarytangles occur with normal aging, but they are much more prevalent inpersons with Alzheimer's disease.

c. Parkinson's Disease.

Parkinson's Disease (PD) is an idiopathic, slowly progressive,degenerative CNS disorder characterized by slow and decreased movement(bradykinesia), muscular rigidity, resting tremor (dystonia), musclefreezing, and postural instability. Originally considered primarily amotor disorder, PD is now recognized to also cause depression andemotional changes. PD also can affect cognition, behavior, sleep,autonomic function, and sensory function. The most common cognitiveimpairments include an impairment in attention and concentration,working memory, executive function, producing language, and visuospatialfunction. A characteristic of PD is symptoms related to reduced motorfunction usually precede those related to cognitive impairment, whichaids in diagnosis of the disease.

In primary Parkinson's disease, the pigmented neurons of the substantianigra, locus coeruleus, and other brain stem dopaminergic cell groupsdegenerate. The cause is not known. The loss of substantia nigraneurons, which project to the caudate nucleus and putamen, results indepletion of the neurotransmitter dopamine in these areas. Onset isgenerally after age 40, with increasing incidence in older age groups.

Parkinson's disease is newly diagnosed in about 60,000 Americans eachyear and currently affects approximately one million Americans. Eventhough PD is not fatal in itself, its complications are the fourteenthleading cause of death in the United States. At present, PD cannot becured, and treatment is generally prescribed to control symptoms, withsurgery prescribed in later, severe cases.

Treatment options for PD include administration of pharmaceuticals tohelp manage motor deficits. These options increase or substitute for theneurotransmitter, dopamine, of which PD patients have low brainconcentrations. Such medications include: carbidopa/levodopa (whichcreate more dopamine in the brain); apomorphine, pramipexolole,ropinirole, and rotingotine (dopamine agonists); selegiline andrasagiline (MAO-B inhibitors which prevent breakdown of dopamine);entacapone and tolcapone (Catechol-O-methyltransferase [COMT] inhibitorswhich make more levodopa available in the brain); benztropine andtrihexyphenidyl (anticholinergics); and amantadine (controls tremor andstiffness). Exercise/physical therapy is also commonly prescribed tohelp maintain physical and mental function.

Current treatment options, however treat the symptoms of PD, are notcurative, and fail to prevent disease progression. Additionally, currentmedications tend to lose efficacy in late stage PD. The most prescribeddrug, levodopa, commonly results in adverse effects within 5 to 10 yearsafter commencing the medication. These adverse effects can be severe andcan result in motor fluctuations and unpredictable swings in motorcontrol between doses as well as jerking/twitching (dyskinesia) whichare difficult to manage and are even as disabling as PD's own symptoms.Thus, there remains a need for new therapies with new mechanisms ofaction which can either be administrated along or in combination withcurrent PD medications.

d. Parkinsonism. Secondary parkinsonism (also referred to as atypicalParkinson's disease or Parkinson's plus) results from loss of orinterference with the action of dopamine in the basal ganglia due toother idiopathic degenerative diseases, drugs, or exogenous toxins. Themost common cause of secondary parkinsonism is ingestion ofantipsychotic drugs or reserpine, which produce parkinsonism by blockingdopamine receptors. Less common causes include carbon monoxide ormanganese poisoning, hydrocephalus, structural lesions (tumors, infarctsaffecting the midbrain or basal ganglia), subdural hematoma, anddegenerative disorders, including nigrostriatal degeneration. Certaindisorders like Progressive Supranuclear Palsy (PSP), Multiple SystemAtrophy (MSA), Corticobasal degeneration (CBD) and Dementia with LewyBodies (DLB) can exhibit Parkinsonism symptoms before the cardinalsymptoms necessary to the specific diagnosis can be made, and thus maybe labeled as “Parkinsonism.”

e. Frontotemporal dementia. Frontotemporal dementia (FTD) is a conditionresulting from the progressive deterioration of the frontal lobe of thebrain. Over time, the degeneration may advance to the temporal lobe.Second only to Alzheimer's disease (AD) in prevalence, FTD accounts for20% of pre-senile dementia cases. Symptoms are classified into threegroups based on the functions of the frontal and temporal lobesaffected:

Behavioral variant FTD (bvFTD), with symptoms include lethargy andaspontaneity on the one hand, and disinhibition on the other;progressive nonfluent aphasia (PNFA), in which a breakdown in speechfluency due to articulation difficulty, phonological and/or syntacticerrors is observed but word comprehension is preserved; and semanticdementia (SD), in which patients remain fluent with normal phonology andsyntax but have increasing difficulty with naming and wordcomprehension. Other cognitive symptoms common to all FTD patientsinclude an impairment in executive function and ability to focus. Othercognitive abilities, including perception, spatial skills, memory andpraxis typically remain intact. FTD can be diagnosed by observation ofreveal frontal lobe and/or anterior temporal lobe atrophy in structuralMRI scans.

A number of forms of FTD exist, any of which may be treated or preventedusing the subject methods and compositions. For example, one form offrontotemporal dementia is Semantic Dementia (SD). SD is characterizedby a loss of semantic memory in both the verbal and non-verbal domains.SD patients often present with the complaint of word-findingdifficulties. Clinical signs include fluent aphasia, anomia, impairedcomprehension of word meaning, and associative visual agnosia (theinability to match semantically related pictures or objects). As thedisease progresses, behavioral and personality changes are often seensimilar to those seen in frontotemporal dementia although cases havebeen described of ‘pure’ semantic dementia with few late behavioralsymptoms. Structural MRI imaging shows a characteristic pattern ofatrophy in the temporal lobes (predominantly on the left), with inferiorgreater than superior involvement and anterior temporal lobe atrophygreater than posterior.

As another example, another form of frontotemporal dementia is Pick'sdisease (PiD, also PcD). A defining characteristic of the disease isbuild-up of tau proteins in neurons, accumulating into silver-staining,spherical aggregations known as “Pick bodies.” Symptoms include loss ofspeech (aphasia) and dementia. Patients with orbitofrontal dysfunctioncan become aggressive and socially inappropriate. They may steal ordemonstrate obsessive or repetitive stereotyped behaviors. Patients withdorsomedial or dorsolateral frontal dysfunction may demonstrate a lackof concern, apathy, or decreased spontaneity. Patients can demonstratean absence of self-monitoring, abnormal self-awareness, and an inabilityto appreciate meaning. Patients with gray matter loss in the bilateralposterolateral orbitofrontal cortex and right anterior insula maydemonstrate changes in eating behaviors, such as a pathologic sweettooth. Patients with more focal gray matter loss in the anterolateralorbitofrontal cortex may develop hyperphagia. While some of the symptomscan initially be alleviated, the disease progresses and patients oftendie within two to ten years.

f. Huntington's disease. Huntington's disease (HD) is a hereditaryprogressive neurodegenerative disorder characterized by the developmentof emotional, behavioral, and psychiatric abnormalities; loss ofintellectual or cognitive functioning; and movement abnormalities (motordisturbances). The classic signs of HD include the development ofchorea—involuntary, rapid, irregular, jerky movements that may affectthe face, arms, legs, or trunk—as well as cognitive decline includingthe gradual loss of thought processing and acquired intellectualabilities. There may be impairment of memory, abstract thinking, andjudgment; improper perceptions of time, place, or identity(disorientation); increased agitation; and personality changes(personality disintegration). Although symptoms typically become evidentduring the fourth or fifth decades of life, the age at onset is variableand ranges from early childhood to late adulthood (e.g., 70s or 80s).

HD is transmitted within families as an autosomal dominant trait. Thedisorder occurs as the result of abnormally long sequences or “repeats”of coded instructions within a gene on chromosome 4 (4p16.3). Theprogressive loss of nervous system function associated with HD resultsfrom loss of neurons in certain areas of the brain, including the basalganglia and cerebral cortex.

g. Amyotrophic lateral sclerosis. Amyotrophic lateral sclerosis (ALS) isa rapidly progressive, invariably fatal, neurological disease thatattacks motor neurons. Muscular weakness and atrophy and signs ofanterior horn cell dysfunction are initially noted most often in thehands and less often in the feet. The site of onset is random, andprogression is asymmetric. Cramps are common and may precede weakness.Rarely, a patient survives 30 years; 50% die within 3 years of onset,20% live 5 years, and 10% live 10 years.

Diagnostic features include onset during middle or late adult life andprogressive, generalized motor involvement without sensoryabnormalities. Nerve conduction velocities are normal until late in thedisease. Recent studies have documented the presentation of cognitiveimpairments as well, particularly a reduction in immediate verbalmemory, visual memory, language, and executive function.

A decrease in cell body area, number of synapses and total synapticlength has been reported in even normal-appearing neurons of the ALSpatients. It has been suggested that when the plasticity of the activezone reaches its limit, a continuing loss of synapses can lead tofunctional impairment. Promoting the formation or new synapses orpreventing synapse loss may maintain neuron function in these patients.

h. Multiple Sclerosis. Multiple Sclerosis (MS) is characterized byvarious symptoms and signs of CNS dysfunction, with remissions andrecurring exacerbations. The most common presenting symptoms areparesthesias in one or more extremities, in the trunk, or on one side ofthe face; weakness or clumsiness of a leg or hand; or visualdisturbances, e.g., partial blindness and pain in one eye (retrobulbaroptic neuritis), dimness of vision, or scotomas. Common cognitiveimpairments include impairments in memory (acquiring, retaining, andretrieving new information), attention and concentration (particularlydivided attention), information processing, executive functions,visuospatial functions, and verbal fluency. Common early symptoms areocular palsy resulting in double vision (diplopia), transient weaknessof one or more extremities, slight stiffness or unusual fatigability ofa limb, minor gait disturbances, difficulty with bladder control,vertigo, and mild emotional disturbances; all indicate scattered CNSinvolvement and often occur months or years before the disease isrecognized. Excess heat may accentuate symptoms and signs.

The course is highly varied, unpredictable, and, in most patients,remittent. At first, months or years of remission may separate episodes,especially when the disease begins with retrobulbar optic neuritis.However, some patients have frequent attacks and are rapidlyincapacitated; for a few the course can be rapidly progressive.

i. Glaucoma. Glaucoma is a common neurodegenerative disease that affectsretinal ganglion cells (RGCs). Evidence supports the existence ofcompartmentalized degeneration programs in synapses and dendrites,including in RGCs. Recent evidence also indicates a correlation betweencognitive impairment in older adults and glaucoma (Yochim B P, et al.Prevalence of cognitive impairment, depression, and anxiety symptomsamong older adults with glaucoma. J Glaucoma. 2012; 21(4):250-254).

j. Myotonic dystrophy. Myotonic dystrophy (DM) is an autosomal dominantmultisystem disorder characterized by dystrophic muscle weakness andmyotonia. The molecular defect is an expanded trinucleotide (CTG) repeatin the 3′ untranslated region of the myotonin protein kinase gene onchromosome 19q. Symptoms can occur at any age, and the range of clinicalseverity is broad. Myotonia is prominent in the hand muscles, and ptosisis common even in mild cases. In severe cases, marked peripheralmuscular weakness occurs, often with cataracts, premature balding,hatchet facies, cardiac arrhythmias, testicular atrophy, and endocrineabnormalities (e.g., diabetes mellitus). Mental retardation is common insevere congenital forms, while an aging-related decline of frontal andtemporal cognitive functions, particularly language and executivefunctions, is observed in milder adult forms of the disorder. Severelyaffected persons die by their early 50s.

k. Dementia. Dementia describes a class of disorders having symptomsaffecting thinking and social abilities severely enough to interferewith daily functioning. Other instances of dementia in addition to thedementia observed in later stages of the aging-associated disordersdiscussed above include vascular dementia, and dementia with Lewybodies, described below.

In vascular dementia, or “multi-infarct dementia”, cognitive impairmentis caused by problems in supply of blood to the brain, typically by aseries of minor strokes, or sometimes, one large stroke preceded orfollowed by other smaller strokes. Vascular lesions can be the result ofdiffuse cerebrovascular disease, such as small vessel disease, or focallesions, or both. Patients suffering from vascular dementia present withcognitive impairment, acutely or subacutely, after an acutecerebrovascular event, after which progressive cognitive decline isobserved. Cognitive impairments are similar to those observed inAlzheimer's disease, including impairments in language, memory, complexvisual processing, or executive function, although the related changesin the brain are not due to AD pathology but to chronic reduced bloodflow in the brain, eventually resulting in dementia. Single photonemission computed tomography (SPECT) and positron emission tomography(PET) neuroimaging may be used to confirm a diagnosis of multi-infarctdementia in conjunction with evaluations involving mental statusexamination.

Dementia with Lewy bodies (DLB, also known under a variety of othernames including Lewy body dementia, diffuse Lewy body disease, corticalLewy body disease, and senile dementia of Lewy type) is a type ofdementia characterized anatomically by the presence of Lewy bodies(clumps of alpha-synuclein and ubiquitin protein) in neurons, detectablein post mortem brain histology. Its primary feature is cognitivedecline, particularly of executive functioning. Alertness and short termmemory will rise and fall.

Persistent or recurring visual hallucinations with vivid and detailedpictures are often an early diagnostic symptom. DLB it is often confusedin its early stages with Alzheimer's disease and/or vascular dementia,although, where Alzheimer's disease usually begins quite gradually, DLBoften has a rapid or acute onset. DLB symptoms also include motorsymptoms similar to those of Parkinson's. DLB is distinguished from thedementia that sometimes occurs in Parkinson's disease by the time framein which dementia symptoms appear relative to Parkinson symptoms.Parkinson's disease with dementia (POD) would be the diagnosis whendementia onset is more than a year after the onset of Parkinson's. DLBis diagnosed when cognitive symptoms begin at the same time or within ayear of Parkinson symptoms.

l. Progressive supranuclear palsy. Progressive supranuclear palsy (PSP)is a brain disorder that causes serious and progressive problems withcontrol of gait and balance, along with complex eye movement andthinking problems. One of the classic signs of the disease is aninability to aim the eyes properly, which occurs because of lesions inthe area of the brain that coordinates eye movements. Some individualsdescribe this effect as a blurring. Affected individuals often showalterations of mood and behavior, including depression and apathy aswell as progressive mild dementia. The disorder's long name indicatesthat the disease begins slowly and continues to get worse (progressive),and causes weakness (palsy) by damaging certain parts of the brain abovepea-sized structures called nuclei that control eye movements(supranuclear). PSP was first described as a distinct disorder in 1964,when three scientists published a paper that distinguished the conditionfrom Parkinson's disease. It is sometimes referred to asSteele-Richardson-Olszewski syndrome, reflecting the combined names ofthe scientists who defined the disorder. Although PSP gets progressivelyworse, no one dies from PSP itself.

m. Ataxia. People with ataxia have problems with coordination becauseparts of the nervous system that control movement and balance areaffected. Ataxia may affect the fingers, hands, arms, legs, body,speech, and eye movements. The word ataxia is often used to describe asymptom of incoordination which can be associated with infections,injuries, other diseases, or degenerative changes in the central nervoussystem. Ataxia is also used to denote a group of specific degenerativediseases of the nervous system called the hereditary and sporadicataxias which are the National Ataxia Foundation's primary emphases.

n. Multiple-system atrophy. Multiple-system atrophy (MSA) is adegenerative neurological disorder. MSA is associated with thedegeneration of nerve cells in specific areas of the brain. This celldegeneration causes problems with movement, balance, and other autonomicfunctions of the body such as bladder control or blood-pressureregulation.

The cause of MSA is unknown and no specific risk factors have beenidentified. Around 55% of cases occur in men, with typical age of onsetin the late 50s to early 60s. MSA often presents with some of the samesymptoms as Parkinson's disease. However, MSA patients generally showminimal if any response to the dopamine medications used forParkinson's.

o. Frailty. Frailty Syndrome (“Frailty”) is a geriatric syndromecharacterized by functional and physical decline including decreasedmobility, muscle weakness, physical slowness, poor endurance, lowphysical activity, malnourishment, and involuntary weight loss. Suchdecline is often accompanied and a consequence of diseases such ascognitive dysfunction and cancer. However, Frailty can occur evenwithout disease. Individuals suffering from Frailty have an increasedrisk of negative prognosis from fractures, accidental falls, disability,comorbidity, and premature mortality. (C. Buigues, et al. Effect of aPrebiotic Formulation on Frailty Syndrome: A Randomized, Double-BlindClinical Trial, Int. J. Mol. Sci. 2016, 17, 932). Additionally,individuals suffering from Frailty have an increased incidence of higherhealth care expenditure. (Id.)

Common symptoms of Frailty can be determined by certain types of tests.For example, unintentional weight loss involves a loss of at least 10lbs. or greater than 5% of body weight in the preceding year; muscleweakness can be determined by reduced grip strength in the lowest 20% atbaseline (adjusted for gender and BMI); physical slowness can be basedon the time needed to walk a distance of 15 feet; poor endurance can bedetermined by the individual's self-reporting of exhaustion; and lowphysical activity can be measured using a standardized questionnaire.(Z. Palace et al., The Frailty Syndrome, Today's Geriatric Medicine7(1), at 18 (2014)).

In some embodiments, the subject methods and compositions find use inslowing the progression of aging-associated cognitive, motor,neuroinflammatory, or other age-related impairment or condition. Inother words, cognitive, motor, neuroinflammatory, or other abilities orconditions in the individual will decline more slowly followingtreatment by the disclosed methods than prior to or in the absence oftreatment by the disclosed methods. In some such instances, the subjectmethods of treatment include measuring the progression of cognitive,motor, neuroinflammation, or other age-related ability or symptomdecline after treatment, and determining that the progression of declineis reduced. In some such instances, the determination is made bycomparing to a reference, e.g., the rate of decline in the individualprior to treatment, e.g., as determined by measuring cognitive, motor,neuroinflammatory, or other age-related abilities or conditions prior attwo or more time points prior to administration of the subject bloodproduct.

The subject methods and compositions also find use in stabilizing thecognitive, motor, neuroinflammatory, or other abilities or conditions ofan individual, e.g., an individual suffering from aging-associatedcognitive decline or an individual at risk of suffering fromaging-associated cognitive decline. For example, the individual maydemonstrate some aging-associated cognitive impairment, and progressionof cognitive impairment observed prior to treatment with the disclosedmethods will be halted following treatment by the disclosed methods. Asanother example, the individual may be at risk for developing anaging-associated cognitive decline (e.g., the individual may be aged 50years old or older, or may have been diagnosed with an aging-associateddisorder), and the cognitive abilities of the individual aresubstantially unchanged, i.e., no cognitive decline can be detected,following treatment by the disclosed methods as compared to prior totreatment with the disclosed methods.

The subject methods and compositions also find use in reducingcognitive, motor, neuroinflammatory, or other age-related impairment inan individual suffering from an aging-associated impairment. In otherwords, the affected ability is improved in the individual followingtreatment by the subject methods. For example, the cognitive or motorability in the individual is increased, e.g., by 2-fold or more, 5-foldor more, 10-fold or more, 15-fold or more, 20-fold or more, 30-fold ormore, or 40-fold or more, including 50-fold or more, 60-fold or more,70-fold or more, 80-fold or more, 90-fold or more, or 100-old or more,following treatment by the subject methods relative to the cognitive ormotor ability that is observed in the individual prior to treatment bythe subject methods.

In some instances, treatment by the subject methods and compositionsrestores the cognitive, motor, or other ability in the individualsuffering from aging-associated cognitive or motor decline, e.g., totheir level when the individual was about 40 years old or less. In otherwords, cognitive or motor impairment is abrogated. Methods of Diagnosingand Monitoring for Improvement

13. In some instances, among the variety of methods to diagnose andmonitor disease progression and improvement in cognitive disease, motorimpairment, neurodegenerative disease, and/or neuroinflammatory diseasethe following types of assessments are used alone or in combination withsubjects suffering from neurodegenerative disease, as desired. Thefollowing types of methods are presented as examples and are not limitedto the recited methods. Any convenient methods to monitor disease may beused in practicing the invention, as desired. Those methods are alsocontemplated by the methods of the invention.

a. General Cognition

Embodiments of the methods of the invention further comprise methods ofmonitoring the effect of a medication or treatment on a subject fortreating cognitive impairment and/or age-related dementia, the methodcomprising comparing cognitive function before and after treatment.Those having ordinary skill in the art recognize that there arewell-known methods of evaluating cognitive function. For example, andnot by way of limitation, the method may comprise evaluation ofcognitive function based on medical history, family history, physicaland neurological examinations by clinicians who specialize dementia andcognitive function, laboratory tests, and neuropsychological assessment.Additional embodiments which are contemplated by the invention include:the assessment of consciousness, such as using the Glasgow Coma Scale(EMV); mental status examination, including the abbreviated mental testscore (AMTS) or mini-mental state examination (MMSE) (Folstein et al.,J. Psychiatr. Res 1975; 12:1289-198); global assessment of higherfunctions; estimation of intracranial pressure such as by fundoscopy. Inone embodiment, monitoring the effect on cognitive impairment and/orage-related dementia includes a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or12-point improvement using the Alzheimer's Disease AssessmentScale-Cognitive Subscale (ADAS-COG).

In one embodiment, examinations of the peripheral nervous system may beused to evaluate cognitive function, including any one of thefollowings: sense of smell, visual fields and acuity, eye movements andpupils (sympathetic and parasympathetic), sensory function of face,strength of facial and shoulder girdle muscles, hearing, taste,pharyngeal movement and reflex, tongue movements, which can be testedindividually (e.g. the visual acuity can be tested by a Snellen chart; areflex hammer used testing reflexes including masseter, biceps andtriceps tendon, knee tendon, ankle jerk and plantar (i.e. Babinskisign); Muscle strength often on the MRC scale 1 to 5; Muscle tone andsigns of rigidity.

b. Parkinson's Disease

Embodiments of the methods of the invention further comprise methods ofmonitoring the effect of a medication or treatment on a subject fortreating motor impairment, the method comprising comparing motorfunction before and after treatment. Those having ordinary skill in theart recognize that there are well-known methods of evaluating motorfunction. For example, and not by way of limitation, the method maycomprise evaluation of motor function based on medical history, familyhistory, physical and neurological examinations by clinicians whospecialize neurodegeneration and motor impairment, laboratory tests, andneurodegenerative assessment. Additional embodiments which arecontemplated by the invention include employment of the rating scalesdiscussed below.

Several rating scales have been utilized for evaluating the progressionof PD. The most widely-used scales include the Unified Parkinson'sDisease Rating Scale (UPDRS, which was introduced in 1987) (J. RehabilRes. Dev., 2012 49(8): 1269-76), and the Hoehn and Yahr scale(Neruology, 1967 17(5): 427-42). Additional scales include the MovementDisorder Society (MDS)'s updated UPDRS scale (MDS-UPDRS) as well as theSchwab and England Activities of Daily Living (ADL) Scale.

The UPDRS scale evaluates 31 items that contributed to three subscales:(1) mentation, behavior, and mood; (2) activities of daily living; and(3) motor examination. The Hoehn and Yahr scale classifies PD into fivestages with discreet substages: 0—no signs of disease; 1—symptoms on oneside only; 1.5—symptoms on one side but also involving neck and spine;2—symptoms on both sides with no balance impairment; 2.5—mild symptomson both sides, with recovery when the ‘pull’ test is given; 3—balanceimpairment with mild to moderate disease; 4—severe disability, butability to walk or stand unassisted; and 5—need a wheelchair orbedridden without assistance. The Schwab and England scale classifies PDinto several percentages (from 100%−complete independent to 10%−totaldependent).

General motor function can be evaluated using widely-used scalesincluding the General Motor Function Scale (GMF). This tests threecomponents: dependence, pain, and insecurity. (Aberg A. C., et al.(2003) Disabil. Rehabil. 2003 May 6; 25(9):462-72.). Motor function canalso be assessed using home-monitoring or wearable sensors. For example:gait (speed of locomotion, variability, leg rigidity) can be sensed withan accelerometer; posture (trunk inclination) by a gyroscope; legmovement by an accelerometer; hand movement by an accelerometer andgyroscope; tremor (amplitude, frequency, duration, asymmetry) by anaccelerometer; falling by an accelerometer; gait freezing by anaccelerometer; dyskinesia by an accelerometer, gyroscope, and inertialsensors; bradykinesia (duration and frequency) by an accelerometer plusgyroscope, and aphasia (pitch) using a microphone. (Pastorino M, et al.,Journal of Physics: Conference Series 450 (2013) 012055).

c. Multiple Sclerosis

In addition to monitoring improvement for symptoms associated withcognition, the progression or improvement of neurodegenerationassociated with multiple sclerosis (MS) can be monitored usingtechniques well-known to those having ordinary skill in the art. By wayof example, and not limitation, monitoring can be performed throughtechniques such as: cerebrospinal fluid (CSF) monitoring; magneticresonance imaging (MRI) to detect lesions and development ofdemyelinating plaques; evoked potential studies; and gait monitoring.

CSF analysis may be performed, for example, through lumbar puncture toobtain pressure, appearance, and CSF content. Normal values typicallyrange as follows: pressure (70-180 mm H20); appearance is clear andcolorless; total protein (15-60 mg/100 mL); IgG is 3-12% of the totalprotein; glucose is 50-80 mg/100 mL; cell count is 0-5 white blood cellsand no red blood cells; chloride (110-125 mEq/L). Abnormal results mayindicate the presence or progression of MS.

MRI is another technique that may be performed to monitor diseaseprogression and improvement. Typical criteria for monitoring MS with MRIinclude the appearance of patchy areas of abnormal white matter incerebral hemisphere and in paraventricular areas, lesions present in thecerebellum and/or brain stem as well as in the cervical or thoracicregions of the spinal cord.

Evoked potentials may be used to monitor the progression and improvementof MS in subjects. Evoked potentials measure slowing of electricalimpulses such as in Visual Evoked Response (VER), Brain Stem AuditoryEvoked Responses (BAER), and Somatosensory Evoked Responses (SSER).Abnormal responses help to indicate that there is a decrease in thespeed of conduction in central sensory pathways.

Gait monitoring can also be used to monitor disease progression andimprovement in MS subjects. MS is often accompanied by an impairment inmobility and an abnormal gait due in part to fatigue. Monitoring may beperformed, for example, with the use of mobile monitoring devices wornby subjects. (Moon, Y., et al., Monitoring gait in multiple sclerosiswith novel wearable motion sensors, PLOS One, 12(2):e0171346 (2017)).

d. Huntington's

In addition to monitoring improvement for symptoms associated withcognition, the progression or improvement of neurodegenerationassociated with Huntington's Disease (HD) can be monitored usingtechniques well-known to those having ordinary skill in the art. By wayof example, and not limitation, monitoring can be performed throughtechniques such as: motor function; behavior; functional assessment; andimaging.

Examples of motor function that may be monitored as an indication ofdisease progression or improvement include chorea and dystonia,rigidity, bradykinesia, oculomotor dysfunction, and gait/balancechanges. Techniques for performing the monitoring of these metrics arewell-known to those having ordinary skill in the art. (See Tang C, etal., Monitoring Huntington's disease progression through preclinical andearly stages, Neurodegener Dis Manag 2(4):421-35 (2012)).

The psychiatric effects of HD present opportunities to monitor diseaseprogression and improvement. For example, psychiatric diagnoses may beperformed in order to determine whether the subject suffers fromdepression, irritability, agitation, anxiety, apathy and psychosis withparanoia. (Id.)

Functional assessment may also be employed to monitor diseaseprogression or improvement. Total functional score techniques have beenreported (Id.), and often declines by one point per year in some HDgroups.

MRI or PET may be employed also to monitor disease progression orimprovement. For example, there is a loss of striatal projection neuronsin HD, and change in number of these neurons may be monitored insubjects. Techniques to determine neuronal change in HD subjects includeimaging Dopamine D2 receptor binding. (Id.)

e. ALS

In addition to monitoring improvement for symptoms associated withcognition, the progression or improvement of neurodegenerationassociated with Amyotrophic Lateral Sclerosis (ALS) can be monitoredusing techniques well-known to those having ordinary skill in the art.By way of example, and not limitation, monitoring can be performedthrough techniques such as: functional assessment; determining musclestrength; measuring respiratory function; measuring lower motor neuron(LMN) loss; and measuring upper motor neuron (UMN) dysfunction.

Functional assessment can be performed using a functional scalewell-known to those having ordinary skill in the art, such as the ALSFunctional Rating Scale (ALSFRS-R), which evaluates symptoms related tobulbar, limb, and respiratory function. The rate of change is useful inpredicting survival as well as disease progression or improvement.Another measure includes the Combined Assessment of Function andSurvival (CAFS), ranking subjects' clinical outcomes by combiningsurvival time with change in ALSFRS-R. (Simon N G, et al., QuantifyingDisease Progression in Amyotrophic Lateral Sclerosis, Ann Neurol76:643-57 (2014)).

Muscle strength may be tested and quantified through use of compositeManual Muscle Testing (MMT) scoring. This entails averaging measuresacquired from several muscle groups using the Medical Research Council(MRC) muscle strength grading scale. (Id.) Hand-held dynamometry (HHD)may also be used, among other techniques. (Id.)

Respiratory function can be performed using portable spirometry units,used to obtain Forced Vital Capacity (FVC) at baseline to predict theprogression or improvement of the disease. Additionally, maximalinspiratory pressure, sniff nasal inspiratory pressure (SNIP), andsupping FVC may be determined and used to monitor diseaseprogression/improvement. (Id.)

Loss in lower motor neurons is another metric which can be utilized tomonitor disease progression or improvement in ALS. TheNeurophysiological Index may be determined by measuring compound muscleaction potentials (CMAPs) on motor nerve conduction studies, of whichparameters include CMAP amplitude and F-wave frequency. (Id. and deCarvalho M, et al., Nerve conduction studies in amyotrophic lateralsclerosis. Muscle Nerve 23:344-352, (2000)). Lower motor neuron unitnumbers (MUNE) may be estimated as well. In MUNE, the number of residualmotor axons supplying a muscle through estimation of the contribution ofindividual motor units to the maximal CMAP response is estimated, andused to determine disease progression or improvement. (Simon N G, etal., supra). Additional techniques for determining loss of LMN includetesting nerve excitability, electrical impedance myography, and usingmuscle ultrasound to detect changes in thickness in muscles. (Id.)

Dysfunction of upper motor neurons is another metric which can beutilized to monitor disease progression or improvement in ALS.Techniques for determining dysfunction include performing MRI or PETscans on the brain and spinal cord, transcranial magnetic stimulation;and determining levels of biomarkers in the cerebrospinal fluid (CSF).

f. Glaucoma

In addition to monitoring improvement for symptoms associated withcognition, the progression or improvement of neurodegenerationassociated with glaucoma can be monitored using techniques well-known tothose having ordinary skill in the art. By way of example, and notlimitation, monitoring can be performed through techniques such as:determining intraocular pressure; assessment of the optic disc or opticnerve head for damage; visual field testing for peripheral vision loss;and imaging of the optic disc and retina for topographic analysis.

g. Progressive Supranuclear Palsy (PSP)

In addition to monitoring improvement for symptoms associated withcognition, the progression or improvement of neurodegenerationassociated with Progressive Supranuclear Palsy (PSP) can be monitoredusing techniques well-known to those having ordinary skill in the art.By way of example, and not limitation, monitoring can be performedthrough techniques such as: functional assessment (activities of dailyliving, or ADL); motor assessment; determination of psychiatricsymptoms; and volumetric and functional magnetic resonance imaging(MRI).

The level of function of a subject in terms of independence, partialdependence upon others, or complete dependence can be useful fordetermining the progression or improvement in the disease. (See Duff, K,et al., Functional impairment in progressive supranuclear palsy,Neurology 80:380-84, (2013)). The Progressive Supranuclear Palsy RatingScale (PSPRS) is a rating scale that comprises twenty-eight metrics insix categories: daily activities (by history); behavior; bulbar, ocularmotor, limb motor and gait/midline. The result is a score ranging from0-100. Six items are graded 0-2 and twenty-two items graded 0-4 for apossible total of 100. The PSPRS scores are practical measures, androbust predictors of patient survival. They are also sensitive todisease progression and useful in monitoring disease progression orimprovement. (Golbe L I, et al., A clinical rating scale for progressivesupranuclear palsy, Brain 130:1552-65, (2007)).

The ADL section from the UPDRS (Unified Parkinson's Disease RatingScale) can also be used to quantify functional activity in subjects withPSP. (Duff K, et al., supra). Similarly, the Schwab & England ActivitiesDaily Living Score (SE-ADL) can be used for evaluate independence. (Id.)Additionally, the motor function sections of the UPDRS are useful as areliable measure for assessing disease progression in PSP patients. Themotor section may contain, for example, 27 different measures forquantifying motor function in PSP patients. Examples of these includeresting tremor, rigidity, finger tapping, posture, and gait). Asubject's disease progression or improvement may also be assessed byperforming a baseline neuropsychological evaluation completed by trainedmedical personnel, the assessment using the Neuropsychiatric Inventory(NPI) to determine the frequency and severity of behavior abnormalities(e.g. delusions, hallucinations, agitation, depression, anxiety,euphoria, apathy, disinhibition, irritability, and aberrant motorbehavior). (Id.)

Functional MRI (fMRI) can be employed to monitor disease progression andimprovement as well. fMRI is a technique using MRI to measure changes inbrain activity in certain regions of the brain, usually based on bloodflow to those regions. Blood flow is considered to correlate with brainregion activation. Patients with neurodegenerative disorders like PSPcan be subjected to physical or mental tests before or during beingscanned in an MRI scanner. By way of example, and not limitation, testscan be a well-established force control paradigm where patients as askedto produce force with the hand most affected by PSP and maximumvoluntary contraction (MVC) is measured by fMRI immediately after thetest takes place. Burciu, R G, et al., Distinct patterns of brainactivity in progressive supranuclear palsy and Parkinson's disease, Mov.Disord. 30(9):1248-58 (2015)).

Volumetric MRI is a technique where MRI scanners determine volumedifferences in regional brain volume. This may be done, for example, bycontrasting different disorders, or by determining differences in volumeof a brain region in a patient over time. Volumetric MRI may be employedto determine disease progression or improvement in neurodegenerativedisorders like PSP. The technique is well-known to those having ordinaryskill in the art. (Messina D, et al., Patterns of brain atrophy inParkinson's disease, progressive supranuclear palsy and multiple systematrophy, Parkinsonism and Related Disorders, 17(3):172-76 (2011)).Examples of cerebral regions which may be measured include, but are notlimited to, intracranial volume, cerebral cortex, cerebellar cortex,thalamus, caudate, putamen, pallidum, hippocampus, amygdala, lateralventricles, third ventricle, fourth ventricle, and brain stem.

h. Neurogenesis

The invention also contemplates treating or improving neurogenesis in asubject with declining or impaired neurogenesis, which may manifestitself, for example, through reduced cognitive or motor function, orthrough association with neuroinflammation. An embodiment of theinvention includes administering, by way of example and not limitation,a blood plasma, a plasma fraction, or a PPF to the subject with reducedor impaired neurogenesis using a Pulsed Dosing treatment regimen.

An embodiment of the invention also contemplates determining the levelof neurogenesis before, during, and/or after administration of the bloodplasma, plasma fraction, or PPF. Noninvasive techniques for evaluatingneurogenesis have been reported. (Tamura Y. et al., J. Neurosci. (2016)36(31):8123-31). Positron emission tomography (PET) used with thetracer, [18F]FLT, in combinations with the BBB transporter inhibitorprobenecid, allows for accumulation of the tracer in neurogenic regionsof the brain. Such imaging allows for an evaluation of neurogenesis inpatients being treated for neurodegenerative disease.

i. Neuroinflammation

The invention also contemplates treating or improving neuroinflammationin a subject with heightened neuroinflammation, which may manifestitself, for example, through reduced cognitive or motor function, orthrough association with reduced neurogenesis or neurodegeneration. Anembodiment of the invention includes administering, by way of exampleand not limitation, a blood plasma, a plasma fraction, or a PPF to thesubject with neuroinflammation using a Pulsed Dosing treatment regimen.

An embodiment of the invention also contemplates determining the levelof neuroinflammation before, during, and/or after administration of theblood plasma, plasma fraction, or PPF. Noninvasive techniques forevaluating neuroinflammation have been reported such as TSPO PositronEmission Tomography (TSPO PET) using ¹¹C-PK11195 and other such tracers.(See Vivash L, et al., J. Nucl. Med. 2016, 57:165-68; and Janssen B, etal., Biochim. et Biophys. Acta, 2016, 425-41, herein incorporated byreference). Invasive techniques for evaluating neuroinflammation includedrawing of cerebrospinal fluid and detecting, for example, expressionlevels of neuroinflammatory markers or factors such as (but not limitedto) prostaglandin E2, cyclooxygenase-2, TNF-alpha, IL-6, IFN-gamma,IL-10, eotaxin, beta-2 microglobulin, VEGF, glial cell line-derivedneurotrophic factor, chiotriosidase-1, MMP-9, CXC motif chemokine 13,terminal complement complex, chitinase-3-like-protein 1, andosteopontin. (See Vinther-Jensen T, et al., Neruol NeurimmunolNeuroinflamm, 2016, 3(6): e287; and Mishra et al., J. Neuroinflamm.,2017, 14:251 herein incorporated by reference).

14. Combination Stem Cell and Pulsed Dosing Therapy

An embodiment of the invention includes treating a subject diagnosedwith a cognitive impairment, impaired motor function, neuroinflammation,or a decline in neurogenesis by administering to the subject aneffective amount of blood plasma or Plasma Fraction in a subject who isundergoing, will undergo, or has received stem cell therapy. Anotherembodiment of the invention includes administering to a subject aneffective amount of blood plasma or Plasma Fraction where the subject isundergoing, will undergo, or has received stem cell therapy, and whereinthe stem cells used in the therapy can be embryonic stem cells,non-embryonic stem cells, induced pluripotent stem cells (iPSCs), cordblood stem cells, amniotic fluid stem cells, and the like.

Stem cell therapy and techniques to perform such therapy are known tothose having ordinary skill in the art. (Andres R H, et al., Brain 2011,134; 1777-89; Daadi M M, et al., Cell Transplant 2013, 22(5):881-92;Horie N, et al., Stem Cells 2011 29(2):doi: 10.1002/stem.584; Thomsen GM, et al., Stem Cells 2018, doi: 10.1002/stem.2825; U.S. patentapplication Ser. Nos. 09/973,198; 12/258,210; 12/596,884; and Ser. No.13/290,439, which are all incorporated herein by reference). Anotherembodiment of the invention includes treating a subject diagnosed withtraumatic spinal cord injury, stroke, retinal disease, Huntington'sdisease, Parkinson's Disease, Alzheimer's Disease, hearing loss, heartdisease, rheumatoid arthritis, severe burns, or is in need of a bonemarrow transplant and who is undergoing, will undergo, or has receivedstem cell therapy, with an effective amount of blood plasma or PlasmaFraction.

15. Methods of Screening Compositions

Also provided are methods of screening compositions for activity intreating cognitive or motor impairment, reducing neuroinflammation, orincreasing neurogenesis. Such methods are contemplated by the inventionand include those methods described in the experimental examples below.Compositions that may be screened by embodiments of the inventioninclude: biological compositions (e.g. proteins, combinations ofproteins, antibodies, small molecule antagonists); Plasma Fractions, orother blood compositions. Results from the methods of screeningcompositions include, but are not limited to: results ofinflammation/inflammatory markers in the hippocampus (e.g. dentategyrus) or other CNS regions; results of cell proliferation in thehippocampus or other CNS regions; cell survival in the hippocampus orother CNS regions; the cell fate (e.g. astrocytes, new neurons) ofproliferating neuroprogenitor cells (NPCs) in the hippocampus or otherCNS regions; and neurogenesis in the hippocampus or other CNS regions.

Additional embodiments of methods of screening compositions for activityin treating cognitive or motor impairment, reducing neuroinflammation,or increasing neurogenesis include determining acute effects ofcompositions on hippocampus inflammation and proliferation, comprising:5-7 consecutive daily doses of BrdU with concurrent 5-7 consecutivedaily administration of the composition being screened or control(Pulsed Dosed) in rodents or another animal model. Up to 10 days (i.e.1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days) after conclusion of pulsed dosingof the composition being screened, the number of cells in the dentategyrus is determined by BrdU staining, and the percent area exhibitingCD-68 staining (an indicator of inflammation) is determined.

Another embodiment of methods of screening compositions for activity intreating cognitive or motor impairment, reducing neuroinflammation, orincreasing neurogenesis include administering BrdU for 5 consecutivedays (once per day) before commencing a Pulsed Dosing regimen of 5-7days of the composition being screened or control in rodents or otheranimal model. Four, five, six, seven, eight, nine, ten, eleven, ortwelve weeks subsequently, hippocampus cell survival is determined asthe number of cells in the dentate gyrus staining with BrdU,neurogenesis is determined as the number of cells in the dentate gyrusstaining with doublecortin (DCX), and cell fate of neuroprogenitor cellsbecoming either astrocytes (associate with aging) or neurons (notassociated with aging) are determined by co-localization of BrdU withGFAP or NeuN markers, respectively.

Another embodiment of methods of screening compositions for activity intreating cognitive or motor impairment, reducing neuroinflammation, orincreasing neurogenesis include administering BrdU and the compositionbeing screened or control concurrently (and daily) for 5-7 days, andsubsequently determining the degree of neurogenesis by DCX staining inthe hippocampus or cell fate of proliferating NPCs as described above.

Another embodiment of methods of screening compositions for activity intreating cognitive or motor impairment, reducing neuroinflammation, orincreasing neurogenesis include administering a Pulsed Dose regimen ofthe composition to be screened or control, and determining improvementin cognitive or motor function in rodents or another animal model asdescribed in the examples below.

16. Reagents, Devices, and Kits

Also provided are reagents, devices, and kits thereof for practicing oneor more of the above-described methods. The subject reagents, devices,and kits thereof may vary greatly.

Reagents and devices of interest include those mentioned above withrespect to the methods of preparing plasma-comprising blood product fortransfusion into a subject in need hereof, for example, anti-coagulants,cryopreservatives, buffers, isotonic solutions, etc.

Kits may also comprise blood collection bags, tubing, needles,centrifugation tubes, and the like. In yet other embodiments, kits asdescribed herein include two or more containers of blood plasma productsuch as plasma protein fraction, such as three or more, four or more,five or more, including six or more containers of blood plasma product.In some instances, the number of distinct containers of blood plasmaproduct in the kit may be 9 or more, 12 or more, 15 or more, 18 or more,21 or more, 24 or more 30 or more, including 36 or more, e.g., 48 ormore. Each container may have associated therewith identifyinginformation which includes various data about the blood plasma productcontained therein, which identifying information may include one or moreof the age of the donor of the blood plasma product, processing detailsregarding the blood plasma product, e.g., whether the plasma product wasprocessed to remove proteins above an average molecule weight (such asdescribed above), blood type details, etc. In some instances, eachcontainer in the kit includes identifying information about the bloodplasma contained therein, and the identifying information includesinformation about the donor age of the blood plasma product, e.g., theidentifying information provides confirming age-related data of theblood plasma product donor (where such identifying information may bethe age of the donor at the time of harvest). In some instances, eachcontainer of the kit contains a blood plasma product from a donor ofsubstantially the same age, i.e., all of the containers include productfrom donors that are substantially the same, if not the same, age. Bysubstantially the same age is meant that the various donors from whichthe blood plasma products of the kits are obtained differ in each, insome instances, by 5 years or less, such as 4 years or less, e.g., 3years or less, including 2 years or less, such as 1 year or less, e.g.,9 months or less, 6 months or less, 3 months or less, including 1 monthor less. The identifying information can be present on any convenientcomponent of the container, such as a label, an RFID chip, etc. Theidentifying information may be human readable, computer readable, etc.,as desired. The containers may have any convenient configuration. Whilethe volume of the containers may vary, in some instances the volumesrange from 10 ml to 5000 mL, such as 25 mL to 2500 mL, e.g., 50 ml to1000 mL, including 100 mL to 500 mL. The containers may be rigid orflexible, and may be fabricated from any convenient material, e.g.,polymeric materials, including medical grade plastic materials. In someinstances, the containers have a bag or pouch configuration. In additionto the containers, such kits may further include administration devices,e.g., as described above. The components of such kits may be provided inany suitable packaging, e.g., a box or analogous structure, configuredto hold the containers and other kit components.

In addition to the above components, the subject kits will furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, portable flash drive, etc., on which the informationhas been recorded. Yet another means that may be present is a websiteaddress which may be used via the internet to access the information ata removed site. Any convenient means may be present in the kits.

17. Exercise

Exercise can be characterized by aerobic or anaerobic activity, and caninvolve high calorie-burning activity and moderate calorie-burningactivity. Exercise may involve strength training (e.g. weight trainingor isometric exercise). Exercise may also involve, for example, running,bicycling, walking, dancing, marching, swimming, yoga, Tai Chi, balanceexercises, leg bends, jumping rope, surfing, rowing, rotating or flexingthe arms or legs, gardening, cleaning, active games such as bowling,aerobics, Pilates, and martial arts.

An exercise regimen may include performing a single exercise at acertain frequency, or a combination of exercises at a certain frequency.The frequency may be one, two, three, four, five, six, or seven timesper week. The frequency may vary from week-to-week. The exercise regimenmay be at the same level of intensity and/or frequency as the subjectpracticed before administration of the compositions of the invention.The exercise regimen may also be at a higher level of intensity and/orfrequency compared to the levels the subject practiced beforeadministration of the compositions of the invention. The exerciseregimen may have been suggested or prescribed by a health or fitnessprofessional, or the exercise regimen may have been initiated by thesubject himself or herself.

18. Experimental Examples

a. Example 1

Clarified young human plasma (young plasma) or a commercially-availablePPF (“PPF1”) was administered to aged immunocompromised mice(NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, “NSG” strain). PPF1 is a PPF withapproximately 88% normal human albumin (in relation to total protein),12% alpha and beta globulins, and no more than 1% gamma globulin asdetermined by electrophoresis. Except where noted, PPF1 is administeredin the examples herein in vivo using a 5% solution (w/v, 50 g/L). Allmice were homogenized across treatment groups according to 4 differentcriteria: home cage nestlet scoring, initial body weight, open fielddistance traveled, and % center time in open field. Following groupdetermination, mice were injected intraperitoneally (IP) withBrdU(5-bromo-2′-deoxyuridine) formulated in PBS (Phosphate bufferedsaline) at a final concentration of 10 mg/mL dosed at 150 mg/kg for 5days. Following this, mice were injected intravenously (IV) 3 timesweekly with 150 μL of PPF1 for 4 weeks. Behavior testing occurred inweeks 5 and 6, where mice received 2 injections per week to avoidinjections during concurrent testing days. Mice were euthanized 24 hoursafter the final IV injection, for a total of 16 injections over a periodof 6 weeks. Two additional cohorts of mice were injected intravenously(IV) for seven consecutive days with 150 μL of either PPF1 or saline(Pulse Dosed). Behavior testing occurred in weeks 5 and 6 at the sametime as the 3 times per week group.

Behavioral assays were analyzed using CleverSys software (Reston, Va.).CleverSys TopScan V3.0 was used to track mouse behavior in the zeromaze, Barnes maze, open field, and Y-maze. Barnes mazes were constructedby CleverSys. The Grip strength meter was designed and produced byColumbus Instruments (Columbus, Ohio). Y-maze and Open field chamberswere constructed according to specifications of San Diego Instruments(San Diego, Calif.). Histological analysis of hippocampal sections wasperformed on Leica (Buffalo Grove, Ill.) imaging microscope modelDM5500B with DCF7000T brightfield/fluorescent color microscope camera.

Behavioral Testing:

FIG. 1A shows that the groups that were Pulse Dosed with PPF1 trendedtowards increased distance traveled in the open field test as comparedto both the saline control group and the group treated three timesweekly with PPF1. This result indicates a trend towards increasedmotility in the Pulse Dosed group. FIG. 1B shows that the groups thatwere Pulse Dosed with PPF1 trended toward increased percentage of timespent in the center of the open field compared to both the salinecontrol group and the group treated three times weekly with PPF1. Thisresult indicates a trend towards decreased anxiety in the Pulse Dosedgroup.

Body Weight:

FIG. 2 charts the effect of PPF1 on body weight. Both PPF1-treatedgroups (via Pulsed Dosing or thrice weekly) exhibited no detrimentaleffects from injection.

Histology:

FIG. 3 reports the number of DCX positively-labeled cells within thegranule layer of the dentate. There was a significant increase inneurogenesis between the Pulsed Dose PPF1-treated group compared to thethrice weekly treated group and saline group. All data shown aremean±s.e.m. ** P<0.01 One-Way ANOVA with Dunnett's multiple comparisonPost-Hoc analysis (n: saline=8, PPF1 Pulsed Dosed=10, PPF1 3×/week=10).FIG. 4 reports the number of BrdU positively-labeled cells within thegranule layer of the dentate gyrus of three separately treated groups ofmice. There was a significant increase in cell survival between thePulsed Dose PPF1-treated group compared to the thrice weekly treatedgroup and saline group. All data shown are mean±s.e.m. ****P<0.0001,*P<0.05 One-Way ANOVA with Dunnett's multiple comparison Post-Hocanalysis (n: saline=8, PPF1 Pulsed=10, PPF1 3×/week=10).

Analysis of hippocampal sections was performed on Leica (Buffalo Grove,Ill.) imaging microscope model DM5500B with DCF7000Tbrightfield/fluorescent color microscope camera. Ki67 staining Abcam(ab15580) at 1:500 and secondary is goat anti rabbit (Alex Fluor 555)(ab150090) at 1:300.

b. Example 2

Clarified young human plasma (YP), old human plasma (OP) or aCommercially-available PPF (“PPF1”) were administered to agedimmunocompromised mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, “NSG”strain). All mice were homogenized across treatment groups according to4 different criteria: home cage nestlet scoring, initial body weight,open field distance traveled, and % center time in open field. Followinggroup determination, mice were injected intraperitoneally (IP) with BrdUformulated in PBS (Phosphate buffered saline) at a final concentrationof 10 mg/mL dosed at 150 mg/kg for 5 days. Following this, mice wereinjected intravenously (IV) either: 1) Three times per week for 6 weeks(“3×/Week”); 2) Three times in one week only (“3×”); 3) 7 days in oneweek with 150 μL of either clarified young human plasma or PPF1. Anadditional group of mice was administered saline pulsed for 7 days IV.The final group of mice received aged human plasma for either 3 times inone week or for 7 days in one week. All mice were sacrificed 6 weeksafter the initiation of young or aged plasma, PPF1 or vehicle dosing.

Histology:

FIG. 5 reports the number of DCX positively-labeled cells within thegranule layer of the dentate gyrus of nine separately treated groups ofmice treated with either young human plasma (YP), old human plasma (OP),PPF1, or saline. PPF1-treated mice either Pulse Dosed or treated thriceweekly both exhibited increased neurogenesis compared to the othergroups. All data shown are mean±s.e.m; *P<0.05, ANOVA with Dunnett'spost-hoc analysis PPF1 (pulsed or 3×/week) treatment and salinetreatment (n: saline=4, PPF1 pulsed=5, PPF1 3×/week=5, PPF1 3×=4, YPpulsed=6, YP 3×/week=6, YP 3×=4, AP pulsed=6, AP 3×=6)

FIG. 6 reports the number of BrdU positively-labeled cells within thegranule layer of the dentate gyrus of nine separately treated groups ofmice treated with either young human plasma (YP), old human plasma (OP),PPF1, or saline. PPF1-treated mice exhibited a significant increase incell survival compared to the other groups, with Pulse-DosedPPF1-treated mice exhibiting a larger significant difference than thriceweekly dosed PPF1-treated mice. All data shown are mean±s.e.m; **P<0.01,*P <0.05, ANOVA with Dunnett's post-hoc analysis PPF1 (pulsed or3×/week) treatment and saline treatment (n: saline=4, PPF1 pulsed=5,PPF1 3×/week=5, PPF1 3×=4, YP pulsed=6, YP 3×/week=6, YP 3×=4, APpulsed=6, AP 3×=6).

c. Example 3

Clarified young human plasma (YP), old human plasma (OP) or acommercially-available PPF (“PPF1”) were administered to agedimmunocompromised mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, “NSG”strain). Mice were treated with 7 daily intravenous (IV) doses in a 1week regimen.

All mice were homogenized across treatment groups according to 4different criteria: home cage nestlet scoring, initial body weight, openfield distance traveled, and % center time in open field. Followinggroup determination, mice were injected intraperitoneally (IP) with BrdUformulated in PBS (Phosphate buffered saline) at a final concentrationof 10 mg/mL dosed at 150 mg/kg for 5 days. All mice were injectedintravenously (IV) for seven consecutive days (referred to as Pulseddosing) with 150 uL of young or aged human plasma, PPF1 or saline. Threeweeks after pulsed dosing was completed, mice were injectedintraperitoneally (IP) with EdU(5-ethynyl-2′-deoxyuridine) formulated inPBS (Phosphate buffered saline) at a final concentration of 10 mg/mLdosed at 30 mg/kg for 5 days. Barnes maze was performed during week 8 (6weeks following the end of pulse dosing).

Behavioral assays were analyzed using CleverSys software (Reston, Va.).CleverSys TopScan V3.0 was used to track mouse behavior in the Barnesmaze. Barnes maze was constructed by CleverSys. Analysis of hippocampalsections was performed on Leica (Buffalo Grove, Ill.) imaging microscopemodel DM5500B with DCF7000T brightfield/fluorescent color microscopecamera.

Behavioral Testing:

FIG. 7 reports the latency to find the target hole per trial per day foreach treatment group as tested by Barnes Maze. PPF1 Pulsed Dosed-treatedmice exhibited significant decrease in trial latency for severalindividual testing sessions, indicating improved cognitive ability. *P<0.05 mean±s.e.m; unpaired t-test (n: saline=13, PPF1=13, AP=14, YP=14).

Histology:

FIG. 8 reports the number of DCX positively-labeled cells within thegranule layer of the dentate gyrus of four separately treated groups ofmice treated with either young human plasma (YP), old human plasma (OP),PPF1, or saline. There were significant increases in neurogenesis inPulsed Dosed PPF1 and Pulse Dosed young human plasma as compared tosaline treatment. All data shown are mean±s.e.m; ****P<0.0001, **P<0.01,One-Way ANOVA with Dunnett's multiple comparison Post-Hoc analysis. (n:saline=14, PPF1=14, AP=14, YP=15)

FIG. 9 reports the number of BrdU labeled cells within the granule layerof the dentate gyrus of mice treated with either young human plasma(YP), old human plasma (OP), PPF1, or saline. There were significantincreases in cell survival in Pulsed Dosed PPF1 and Pulse Dosed YP ascompared to saline treatment. All data shown are mean±s.e.m;****P<0.0001; mean±s.e.m; One-Way ANOVA with Dunnett's multiplecomparison Post-Hoc analysis. (n: saline=14, PPF1=14, AP=14, YP=15).

d. Example 4

A Commercially-available PPF (“PPF1”) was administered to agedimmunocompromised mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, “NSG”strain). Twelve-month-old mice were treated with a 7-daily tail veinintravenous (IV) doses in 1 week regimen. After treatment, the mice wereallowed to remain in their home cage environment for 4.5 weeks prior tobehavior testing. All injections and behavioral testing took place overthe course of 7 weeks for each cohort and conducted over a total span of9 weeks. All mice received BrdU IP for 5 days prior to first dosing.Mice were sacrificed one day following the conclusion of the lastbehavior test.

Behavioral assays were analyzed using CleverSys software (Reston, Va.).CleverSys TopScan V3.0 was used to track mouse behavior in the Y-maze.

Behavioral Testing:

FIG. 10 reports the percent of total number of entries made into eitherthe familiar or novel arm of total entries made into each arm bytreatment group in the Y-maze test. Twelve-month-old mice were PulseDose treated with saline, PPF1, or 5× concentrated PPF1. PPF1 and PPF1(5×) Pulse Dose treated mice both showed a significant increase inentering the novel arm compared to the amount of entries into the novelarm by saline treated mice, indicating an improvement in cognition. Alldata shown are mean±s.e.m. *P<0.05, paired t-test.

FIG. 11 reports the ratio of bouts into the novel versus the familiararm of the Y-maze for each treatment group. Twelve-month-old mice werePulse Dose treated with saline, PPF1, or 5× concentrated PPF1. PPF1 andPPF1 (5×) Pulse Dose treated mice both exhibited a trend in increasedentry into the novel arm compared to saline treated mice. All data shownare mean±s.e.m.

Histology:

FIG. 12 reports the number of BrdU positively-labeled cells within allhippocampal sections. PPF1 Pulse Dosed mice exhibited a trend forincreased cell survival compared to saline and PPF1 (5×) treated mice.All data shown are mean±s.e.m.

FIG. 13 reports the number of DCX positively-labeled cells within allhippocampal sections. PPF1 and PPF1 (5×) Pulse Dosed mice exhibited atrend for increased neurogenesis compared to saline treated mice. Alldata shown are mean±s.e.m.

e. Example 5

Commercially-available PPF (“PPF1”) was administered to aged(10.5-month-old) immunocompromised mice (NOD.Cg-Prkdscid Il2rgtm 1Wj1/SzJ, “NSG” strain). All mice were homogenized across treatmentgroups according to four different criteria: home cage nestlet scoring,initial body weight, open field distance traveled, and percent centertime in open field. Following group determination, mice were injectedintraperitoneally (IP) with BrdU formulated in PBS (Phosphate bufferedsaline) at a final concentration of 10 mg/mL dosed at 150 mg/kg for 5days. Following this, mice were injected PPF1 intravenously (IV) foreither: 1) 5 sequential days [PPF1-5d] 2) 7 sequential days [PPF1-7d] 3)5 sequential days with an additional 5 sequential days of boosted (B)dosing occurring 6 weeks after the completion of the initial dosing[PPF1-5d-B] 4) 7 sequential days with an additional 7 sequential days ofboosted (B) dosing occurring 6 weeks after the completion of the initialdosing [PPF1-7d-B]. An additional group were injected with saline for 7sequential days with an additional 7 sequential days of dosing occurring6 weeks after the completion of the initial dosing [SAL-7d-B]. Fiveweeks after pulsed dosing, mice were injected IP with EdU(5-ethynyl-2′-deoxyuridine) formulated in PBS at a final concentrationof 10 mg/mL dosed at 30 mg/kg for 5 days. All mice were sacrificed 12weeks after the completion of pulse dosing PPF1 or vehicle.

Analysis of hippocampal sections was performed on Leica (Buffalo Grove,Ill.) imaging microscope model DM5500B with DCF7000Tbrightfield/fluorescent color microscope camera. FIG. 14 reports thenumber of DCX positively labeled cells within the granule layer of thedentate gyrus in PPF1 and saline-treated animals. These results showthat there is a significant improvement in the group treated for 5sequential days followed by a booster, which is comparable to the grouptreated for 7 sequential days. All data shown are mean±s.e.m; PPF1-7d,PPF1-5d-B vs. saline *P<0.05, ANOVA with Dunnett's post-hoc analysis (n:saline=5, PPF1-5d=8, PPF1-7d=7, PPF1-5d-B=8, PPF1-7d-B=7).

FIG. 15 reports the number of BrdU positively labeled cells within thegranule layer of the dentate gyrus in PPF1 and saline-treated animals.These results show that in terms of proliferating cells, inducementincreases in earnest in the group treated 5 days sequentially followedby a booster, compared to the groups treated with 5 or 7 sequential dayswithout a booster. Additionally, booster treatment significantlyincreases cell survival overall. All data shown are mean±s.e.mPPF1-5d-B, PPF1-7d-B vs. saline ***, P<0.001, *P<0.05, ANOVA withDunnett's post-hoc analysis. PPF1-5d vs. PPF1-5d-B +P<0.05, UnpairedT-Test. (n: saline=7, PPF1-5d=8, PPF1-7d=7, PPF1-5d-B=8, PPF1-7d-B=7).

FIG. 16 reports the number of EdU positively labeled cells within thegranule layer of the dentate gyrus in young plasma, PPF1 andsaline-treated animals. These results show that the effects observedwith booster dosing are not due to an increase in the total number ofproliferating cells present, but to an enhanced survival mechanismelicited by booster administration. All data shown are mean±s.e.m; (n:saline=4, PPF1-5d=7, PPF1-7d=6, PPF1-5d-B=7, PPF1-7d-B=6).

f. Example 6

Commercially-available PPF (“PPF1”) was administered to adult (3 and6-month-old) immunocompromised mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ,“NSG” strain). All mice were homogenized across treatment groupsaccording to four different criteria: home cage nestlet scoring, initialbody weight, open field distance traveled, and % center time in openfield. Following group determination, mice were injectedintraperitoneally (IP) with BrdU formulated in PBS (Phosphate bufferedsaline) at a final concentration of 10 mg/mL dosed at 150 mg/kg for 5days. Following this, mice were injected with either saline or PPF1intravenously (IV) for 7 sequential days (pulse dosing). A subset ofmice from both saline and PPF1 treatments were provided running wheelsin their home cage. Mice were sacrificed either 3 days, 10 days or 42days post completion of pulse dosing.

FIG. 17 reports the number of DCX positively labeled cells within thegranule layer of the dentate gyrus in 3-month-old NSG animals treatedwith PPF1 or saline-treatment with or without running wheels. All datashown are mean±s.e.m; Running wheel+PPF1 42d post, Running wheel 42dpost vs. saline 42d post ****P<0.0001, *P<0.05, ANOVA with Dunnett'spost-hoc analysis. Running wheel vs. PPF1 42d post +++P<0.001, Unpairedt-test. (n: saline 3d post=8, PPF1 3d post=8, PPF1 10d post=7, Vehicle42d post=8, PPF1 42d post=8, Running wheel 42d post=8, Runningwheel+PPF1 42d post=8). FIG. 17 also reports the number of DCXpositively labeled cells within the granule layer of the dentate gyrusin 6-month-old NSG animals treated with PPF1 or saline-treatment with orwithout running wheels. All data shown are mean±s.e.m; Runningwheel+PPF1 42d post, Running wheel 42d post vs. saline 42d post****P<0.0001, **P<0.01, ANOVA with Dunnett's post-hoc analysis. Runningwheel vs. PPF1 42d post +++P<0.001, Unpaired t-test. PPF1 42d post vs.saline 42d post +P<0.05, Unpaired t-test. (n: saline 3d post=7, PPF1 3dpost=8, PPF1 10d post=6, saline 42d post=8, PPF1 42d post=6, Runningwheel 42d post=8, Running wheel+PPF1 42d post=9).

FIG. 18 reports the number of Ki67 positively labeled cells within thegranule layer of the dentate gyrus in 3-month-old NSG animals treatedwith PPF1 or saline-treatment with or without running wheels. All datashown are mean±s.e.m; Running wheel+PPF1 42d vs. saline 42d post***P<0.001, ANOVA with Dunnett's post-hoc analysis. (n: saline 3dpost=6, PPF1 3d post=6, PPF1 10d post=7, saline 42d post=8, PPF1 42dpost=8, Running wheel 42d post=8, Running wheel+PPF1 42d post=8).

FIG. 18 also reports the number of Ki67 positively labeled cells withinthe granule layer of the dentate gyrus in 6-month-old NSG animalstreated with PPF1 or saline-treatment with or without running wheels.All data shown are mean±s.e.m; Running wheel+PPF1 42d post, Runningwheel 42d post vs. saline 42d post ***P<0.001, *P<0.05, ANOVA withDunnett's post-hoc analysis (n: saline 3d post=7, PPF1 3d post=7, PPF110d post=8, saline 42d post=8, PPF1 42d post=7, Running wheel 42dpost=7, Running wheel+PPF1 42d post=9).

FIG. 19 reports the number of BrdU positively labeled cells within thegranule layer of the dentate gyrus in 3-month and 6-month-old NSGanimals treated with PPF1 or saline-treatment with or without runningwheels. All data shown are mean±s.e.m; Running wheel+PPF1 42d vs.Vehicle 42d post ***P<0.001, ANOVA with Dunnett's post-hoc analysis.(****P<0.0001; ***P<0.001; **P<0.01; *P<0.05, ANOVA with Dunnett'spost-hoc analysis).

These results show that there is significant enhancement in neurogenesiswith PPF1 and running wheel compared to vehicle 6 weeks post-dosing in3-month-old NSG mice. Additionally, there is significant enhancement inneurogenesis with PPF1 and running wheel compared to running wheelalone, 6 weeks post dosing in 3-month-old NSG mice. There is alsosignificant enhancement in neurogenesis with PPF1 and running wheelcompared to vehicle, 6 weeks post dosing in 6 mo old NSG mice. Theseresults also show significant enhancement in neurogenesis with PPF1 andrunning wheel compared to running wheel alone, 6 weeks post dosing in6-month-old NSG mice. Further there is significant enhancement inprogenitor cell proliferation with PPF1 and running wheel compared tovehicle, 6 weeks post dosing in both 3-month-old and 6-month-old NSGmice.

These findings in adult NSG mice at 6 months of age indicate potentialsynergistic effects with exercise and PPF1 administration which resultsin significant enhancement of neurogenesis as compared to eitherexercise or PPF1 treatments separately. This supports potential utilityof PPF1 treatment in conjunction with an exercise regimen in clinicalsettings. Additionally, these data demonstrate that there is significantcapacity for neurogenesis in the brain that can be accessed via multipleindependent or overlapping mechanisms.

g. Example 7

PPF1 or saline control were administered to two treatment groups of11-month-old immunocompromised mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ,“NSG” strain). All mice received IV injections of 150 μL of PPF1 orsaline per dose for seven consecutive days. A running wheel(MedAssociates) was placed in the cages of the mice designated asrunners (n=8, n=8 for PPF1 and saline) starting on week 7 of the study.The number of wheel revolutions was recorded for 5 consecutive days, dayand night.

FIG. 20 reports the number of wheel revolutions during given timeperiods, with shaded areas indicating a dark cycle. An unpaired t-testwas used to assess statistical significance of total running for bothtreated and untreated groups in the light and dark cycles. Rhythmicexpression profiles were extracted and characterized using time andfrequency domain analysis for a 13-time point series, separately foreach mouse from treated and untreated groups with five 13-time pointseries per mice. Period, phase and amplitude were the parameters definedfor each rhythm and were compared between the two groups using unpairedtwo-sided t-test. Mice treated with PPF1 ran significantly more thanuntreated animals, an indicator of improved motor activity. Mice weresubjected to a hot plate test to control for normal pain sensation intheir paws. Loss of sensation could have affected prior behavioralreadouts. Hot plate testing led to a slight increase in activity afterreturning to the running wheel cage environment as evident by the spikein wheel revolutions indicated in the boxed segment of FIG. 20.

h. Example 8

Recombinant human albumin (“rhAlbumin,” Albumedix, Ltd, Nottingham, UK),clarified young human plasma (“YP”), or saline control were administeredto 10.5-month-old immunocompromised mice (NOD.Cg-Prkdscid Il2rgtm 1Wj1/SzJ, “NSG” strain). All animals received 50 mg/kg of BrdU IP in week1 prior to 7-day pulse dosing. rhAlbumin and YP were diluted to 50 mg/mLin water for injections (WFI, 0.9% saline). All mice received IVinjections of 150 μL of rhAlbumin, YP, or saline per dose for 7consecutive days. Mice were sacrificed 6 weeks after the last day oftreatment.

FIG. 21A shows the amount of cell survival in all 3 treatment groups asdetermined by the number BrdU-labeled cells in the dentate gyrus (“DG”).Young plasma significantly increased cell survival compared to salineand rhAlbumin, whereas rhAlbumin had no significant effect on cellsurvival. All data shown are mean±s.e.m. (***P<0.001 by unpairedt-test).

FIG. 21B shows the amount of DCX staining in all 3 treatment groups asdetermined by the number of DCX positive cells in the dentate gyrus(“DG”). Young plasma significantly increased neurogenesis compared tosaline and rhAlbumin, whereas rhAlbumin was associated with a decreasein neurogenesis as compared to saline control. All data shown aremean±s.e.m. (**P<0.01; ***P<0.001 by unpaired t-test).

i. Example 9

Dissociated mixed neuronal cells derived from mouse E16 cortex wereplated and grown on a 48-well multielectrode array plate (AxionBiosystems). Each well contain 16 electrodes which are in physicalcontact with the plated neuronal cells and measure subtle changes in thecellular membrane properties. This setup allows assessing a variety ofdifferent parameters to get information about neuronal spiking activityand firing behavior at single electrode level, as well as informationabout the extent of neuronal connectivity by assessing synchrony of theneuronal firing properties across multiple electrodes within a well.

The neuronal cultures were maintained in the presence of the treatmentconditions from day 1 onwards. Treatment conditions comprised Neurobasalmedium plus B27 supplements containing 10% (v/v): recombinant humanAlbumin ((“rhAlbumin,” Albumedix, Ltd, Nottingham, UK); PPF1; or HAS1.PBS constituted the control. Neuronal activity was measured at day 7 andday 14 in culture.

FIG. 22 shows that 7 days of PPF1 treatment leads to an increase in theneuronal network activity in comparison to control, rhAlbumin, or HAS1treatment. HAS1 is a commercially-available HAS with over 95% humanalbumin (in relation to total protein) in a 5% solution (w/v, 50 g/L),prepared by a cold alcohol fractionation method, and derived from pooledhuman plasma from donors. Both PPF1 and HAS1 come in a 5% solution(w/v/, 50 g/L) and were diluted 1:10 in Neurobasal medium plus B27supplements. The effect of PPF1 on neuronal network activity persiststhrough to 14 days in culture. This indicates that PPF1 is associatedwith promotion of neuronal network maturation. Data shown as mean±s.e.m.(*P<0.05 by unpaired t-test).

j. Example 10

Clarified old human plasma (OP) or sterile saline were administered to8-week-old (young) immunocompromised mice (NOD.Cg-Prkdscid Il2rgtm 1Wj1/SzJ, “NSG” strain). In each experiment mice were homogenized acrosstreatment groups by weight. All mice were injected IP on 5 consecutivedays with 150 mg/kg of BrdU in sterile PBS. BrdU injection was followedby IV administration of old plasma in different treatment paradigms at150 μL per dose. All paradigms are outlined in FIG. 23.

Paradigm 1 involves twice weekly injections for a total of 10 injectionsover 5 weeks. Histological analysis was performed 48 hours after thelast plasma dose. Paradigm 2 involves thrice weekly injections for atotal of 10 injections over 4 weeks, with histological analysis 48 hoursafter the last dose. In Paradigm 3 mice were injected daily for 7consecutive days and analyzed histologically 48 hours after the lastdose. In Paradigm 4, mice were injected daily for 7 consecutive days andanalyzed 21 days after the last dose. The brains of old plasma treatedmice were analyzed for a marker of endothelial inflammation, VCAM-1 inhippocampus, and for the number of newborn neurons as marked bydoublecortin (DCX) positive cells in the dentate gyrus. VCAM-1 wasimaged on a Hamamatsu NanoZoomer HT (Hamamatsu) afterimmunohistochemistry on 30 μm free floating sections and analyzed usingImage Pro Software (Media Cybernetics). DCX positive cells in thedentate gyrus were counted live on a Leica wide field microscope(Leica).

Analysis of the percent VCAM-1 positive area in the hippocampus (FIGS.24A-24D) shows that endothelial inflammation is significantly increased48 hours after the last plasma administration, with a trend at twiceweekly dosing (FIG. 24A) and significant increases after thrice weekly(FIG. 24B) and Pulsed Dosing (FIG. 24C). VCAM-1 levels were no longersignificantly enhanced 21 days after the last plasma dose wasadministered (FIG. 24D).

Effects on doublecortin were only possible to observe after a 3-4 weektime period, so the number of DCX positive cells were analyzed in thedentate gyrus in paradigms 1, 2 and 4. Analysis revealed that there wasno effect of old plasma on neurogenesis with twice weekly (FIG. 25A) orthrice weekly (FIG. 25B) dosing paradigms, however pulsed dosing for 7consecutive days (FIG. 25C) resulted in a significant decrease in thenumber of DCX positive cells. This data suggests that only pulsed dosingof old human plasma had significant effects on neurogenesis.

k. Example 11

Eight-week-old NSG mice treated for 7 consecutive days with old humanplasma (65-68-year-old origin) were tested using the Modified BarnesMaze 4 weeks after the last injection old plasma. FIG. 26 shows theBarnes Maze escape latency time course and reports the time to reach andenter the escape hole for old plasma and saline-treated NSG mice. Therewere no significant differences in escape latency between groups, but onday 4 old plasma treated mice performed less well than the salinecontrols. This data indicates reduced learning and memory in a spatialmemory task associated with hippocampal function. All data shown aremean±s.e.m. Two-way ANOVA, Sidak post-hoc test).

FIG. 27 depicts the average escape latency in the last three Barnes Mazetrials on day 4. Old plasma treated mice showed a trend towards higherescape latency indicative of impaired memory function. All data shownare mean±s.e.m. (unpaired t-test).

FIG. 28 depicts the difference in escape latency between Barnes Mazetrials 1 and 3 and shows that these trials can be used as a measure oflearning within a single day. Old plasma treated mice have asignificantly lower difference in escape latency between these trialsrevealing decreased learning ability. All data shown are mean±s.e.m.(*P<0.05 by unpaired t-test).

FIG. 29 reports the results of qPCR which was used to quantify mRNAlevels of different markers associated with neurogenesis and synapticfunction. Relative expression levels of doublecortin (DCX), a marker fornewborn neurons, was decreased in agreement with histological analysisof the same marker. In addition, there were trends towards decreasedlevels of vglut1 (vesicular glutamate transporter 1), a marker ofglutamatergic synapses, synaptic marker syn1 (synapsin 1), tuj1 (betaIII tubulin), and bdnf (brain-derived neurotrophic factor). Thesedecreases indicate an overall impaired synaptic and neuronal network inthe brains of old plasma-injected mice. All data shown are mean±s.e.m.(*P<0.05 by unpaired t-test).

l. Example 12

Young (8-week-old) immunocompromised mice (NOD.Cg-Prkdscid Il2rgtm 1Wj1/SzJ, “NSG” strain) were homogenized across treatment groups byweight. Animals were injected subcutaneously (s.c) with 35 mg/kg ofKainic acid (Sigma) in sterile saline or saline control. PeripheralKainic acid administration resulted in acute seizure activity,inflammation in the hippocampus and in a subset of mice also in neuronalloss in the CA1 region of the hippocampus. Two hours after Kainic acidinjection, mice were intravenously dosed with 150 μl of PPF1 or saline.Administration of PPF1 or saline was continued daily for a total of 5days (FIG. 30). Tissue was collected for analysis on day 6. Inflammatorychanges in the CA1 region of the hippocampus were analyzed afterimmunofluorescent staining for microglial activation (CD68) andastrocyte activation (GFAP). Sections were imaged on a HamamatsuNanoZoomer HT (Hamamatsu) after immunohistochemistry on 30 μm freefloating sections and analyzed using Image Pro Software (MediaCybernetics).

Analysis of the percent CD68 positive area in the CA1 region of thehippocampus shows that Kainic acid administration results in increasedCD68 immunoreactivity suggesting increased microglial activation (FIG.31A). Five days of PPF1 administration results in a significant decreaseof the percentage of CD68 positive area and therefore a reduction inmicroglial activation. Similarly, analysis of the percentage of GFAPpositive area (FIG. 31B) shows a significant increase after Kainic acidadministration, which is significantly reduced after PPF1 dosing. Thedata suggests that PPF1 has an acute anti-inflammatory effect in thebrains of mice that have been dosed with Kainic acid. *P<0.05 One-WayANOVA with Dunnett's multiple comparison Post-Hoc analysis.

m. Example 13

NSG mice at 6 months of age were injected daily for one week (7 days),IV, with either PPF1 or saline control at a dose of 150 μL (10 mg/mL).All mice were treated with BrdU 50 mg/kg of BrdU IP once per day on thesame days they received PPF1 or saline control. The mice were thendivided into two cohorts. The first cohort was sacrificed one dayimmediately after the 7 days of concurrent treatment with BrdU and PPF1.The second cohort was sacrificed 7 days later, and received anadditional 7 days of daily BrdU administration.

FIG. 32 shows the number of cells stained in the dentate gyrus ofcohorts 1 and 2 (left to right). Both cohorts exhibited increased cellproliferation in the dentate gyrus compared to saline control. (***p<0.001 unpaired t-test).

n. Example 14

NSG mice at either 3 or 6 months of age were injected daily for one week(7 days), IV, with either PPF1 or saline vehicle. Mice were subsequentlysacrificed 3, 10, or 42 days after the 7 daily doses were administered.Brains were stained with Ki67, a nuclear marker only present inproliferating cells which marks neural stem and progenitor cells in theblade of the dentate gyrus. FIG. 33 shows that 6-month-old miceexhibited an increase in total progenitor cells (Ki67 positive or“Ki67+”) in the dentate gyrus at 10 days following the termination ofthe 7-consecutive day pulse dosing regimen using PPF1. FIG. 34 shows thestaining (bright areas) of Ki67 in the dentate gyrus at 10 days in NSGmice following the termination of the 7-consecutive day pulse dosingregimen using PPF1. This shows that one possible mechanism of action forPPF1 in increasing total cell survival and neurogenesis at 42 daysfollowing cessation of dosing could be due to an increase in totalprogenitor cells (neural stem cells).

o. Example 15

Commercially-available PPF (“PPF1”) or saline control was administeredto two different populations of 6 and 12-month-old immunocompromisedmice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, “NSG” strain). All animalsreceived 50 mg/kg of BrdU in week 1 prior to 7-day pulse dosing of testagent. All mice received IV injections of 150 μL of PPF1 or saline perdose for 7 consecutive days. One cohort from each treatment group wasused to investigate proliferation and was sacrificed 6 weeks after thelast administered dose.

FIG. 35A reports that the cohort of 6-month-old mice treated with PPF1exhibited a significant increase in the number of progenitor cellsdifferentiated into neurons (NeuN+) compared to saline control, and areduction in the number of progenitor cells differentiated intoastrocytes (GFAP+) compared to control. All data shown are mean±s.e.m.(**P<0.01 by unpaired t-test).

FIG. 35B reports that the cohort of 12-month-old mice treated with PPF1exhibited a significant increase in the number of progenitor cellsdifferentiated into neurons (NeuN+) compared to saline control, and astatistically-insignificant difference in the number progenitor cellsdifferentiated into of astrocytes compared to control. All data shownare mean±s.e.m. (**P<0.01 by unpaired t-test).

p. Example 16

Clarified old human plasma (old plasma) or sterile saline wereadministered to 3-month-old mice (NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ,“NSG” strain). In each experiment mice were homogenized across treatmentgroups by weight. All mice were injected IP on 5 consecutive days with150 mg/kg of BrdU in sterile saline. BrdU injection was followed by IVadministration of old plasma or sterile saline at 150 μL per dose dailyfor 7 consecutive days and analyzed histologically 4 weeks after thelast dose.

FIGS. 36A and 36B depict the cell fate of BrdU-labeled proliferatingneural progenitor cells 4 weeks after the last dose. In mice injectedwith old plasma, surviving BrdU-labeled cells differentiatesignificantly less into neurons than into astrocytes. This indicatesthat old human plasma changes the cell fate of neural progenitor cellsin young mice towards the astrocyte lineage (FIG. 36B) and negativelyimpacts the number of newborn neurons in the dentate gyrus (FIG. 36A)(n=12 per group). All data shown are mean±s.e.m. (***P<0.001;****P<0.0001 by unpaired t-test).

q. Example 17

Cortical Activation.

Aged (18 months old) C57BL/6 mice received daily IV injections of 150 ulPPF1 or 0.9% sterile saline for 7 days. Two and a half (2.5) hours afterthe last test agent administration, mice were sacrificed by transcardialperfusion with 0.9% saline followed by 4% formaldehyde under deepanesthesia with ketamine and xylazine. The brains were dissected,post-fixed and then processed with the iDisco procedure to visualizecFos positive cells via Light Sheet Fluorescence Microcopy (LSFM) at2×2×3 micrometer voxel resolution. The imaged brains were aligned as 3Dvolumes and activated cFos positive cells were computationally detected.The statistical comparison between groups was performed by negativebinomial regression corrected for multiple comparisons by falsediscovery rate. (* indicates a q-value of less than 0.05).

Analysis of the mouse brains showed an overall increase in the number ofcFos positive cells in the whole brain volume as well as in cortex andisocortex in PPF1 treated 18-month-old mice (FIGS. 37A-37C). Using thebinomial regression corrected for multiple comparison the differencesthese increases in overall positive cFos numbers did not reachsignificance. However, analysis of more defined cortical areas, such asthe frontal, orbital, infralimbic and prelimbic cortex showed asignificant elevation in the number of cFos positive cells (FIGS.38A-38D) indicative of increased neuronal activity. Enhanced activity inthe pre-frontal cortex area is correlated with enhanced cognitiveperformance, suggesting that PPF1 treatment results in cognitiveimprovements in aged C57BL/6 mice. Similar significant increases in cFospositive cell numbers were also found in the accessory olfactory nucleusand the olfactory tubercle (FIGS. 39A-39B). These areas are associatedwith processing of olfactory information and the enhancement in activitysuggests increased olfactory function. Voxel statistics-basedvisualization of the cFos activation in red showed the increase of cFossignal in the cortex of mice treated with PPF1 (FIG. 40).

r. Example 18

Commercially-available PPF (“PPF1”) or saline control was administeredto 22-month-old wild type (WT) mice (C57BL/6J, “WT”, Strain Code 0664,Jackson Labs, Bar Harbor, Me.). All animals received 50 mg/kg of BrdU inweek 1 prior to 7-day pulse dosing. Subsequently, all mice received IVinjections of 150 μL of PPF1 or saline per dose for seven consecutivedays. Mice were sacrificed 10 days after the last PPF1 or salineinjection and the brains were processed for histology.

FIG. 41A reports the percent CD68 immunoreactive area in the hippocampus(n=10, 10). FIG. 41B reports the percent Iba-1 immunoreactive area inthe hippocampus (n=10, 10). FIG. 41C reports the percent GFAPimmunoreactive area in the hippocampus (n=10, 10). All data shown aremean±s.e.m. (*P<0.05; **P<0.01 by unpaired t-test). These results show asignificant decrease in the microglial markers, CD68 and Iba-1 in thehippocampus of PPF1-treated old mice.

s. Example 19

Commercially-available PPF (“PPF1”) or saline control was administeredto 23-month-old wild type (C57BL/6J, “WT”, Strain Code 0664, JacksonLabs, Bar Harbor, Me.). All animals received 50 mg/kg of BrdU in week 1prior to seven consecutive day pulse dosing. Subsequently, all micereceived IV injections of 150 μL of PPF1 or saline per dose for sevenconsecutive days. One cohort from each treatment group was used toinvestigate histological markers for neuroinflammation and wassacrificed 6 weeks after the last administered dose.

FIG. 42A reports the percent change in BrdU expression compared tosaline control at 6, 9, and 12 weeks post-dosing, which is an indicatorof cell survival in the hippocampus. Animals were treated with a7-consecutive day Pulsed Dosing regimen.

FIG. 42B reports the percent change in doublecortin (DCX) expressioncompared to saline control at 6, 9, and 12 weeks post-dosing, which isan indicator of neurogenesis in the hippocampus. Animals were treatedwith a 7-consecutive day Pulsed Dosing regimen.

t. Example 20

Thirty (30) male alpha-synuclein transgenic mice (Line 61, wild typebackground C57BL/6J), aged 4 to 4.5 month-old, were divided into twogroups of 15 and treated with either PPF1 or vehicle for seven (7)consecutive days. PPF1 treatment was administered IV at 5 μL per gram ofbody weight. Alpha-synuclein mice serve as a transgenic model forParkinson's Disease and over-expresses the alpha-synuclein protein. Thistransgenic model is not immunocompromised, unlike NSG mice.

One day after the last treatment of PPF1 or vehicle, all mice weresubjected to behavior and motor function testing such as nest building,pasta gnawing, wire suspension, rota-rod and beam walk. Pasta gnawing,wire suspension, and beam walk were executed a second time at the end ofthe study. Testing was performed in a randomized order.

Animals were weighed once weekly. FIGS. 43A and 43B show that there wereno significant differences between the PPF1 and vehicle-treated (veh)alpha-synuclein transgenic mice (“Tg”).

FIG. 44 reports the results from nest building. Mice were housedindividually in cages containing wood chip bedding and one square ofpressed cotton (“nestlet”). No other nesting material (e.g. wood wool)was present. The nestlet was introduced on the day before the evaluationof the nest status in about 2 to 3 hours before the dark phase wasinitiated and the next building behavior was evaluated on the followingday of the experiment within about 2 to 3 hours after the light phasestarted. The time span between introduction of the cotton square andevaluation of the next status was the same for all examinations. Themanipulation of the nestlet and the constitution of the built nest wereassessed, according to a five-point scale (Deacon, R M 2006, Assessingnest building in mice. Nat Protoc 1:1117-19.) As shown in FIG. 44, therewas an increased trend in nesting behavior in PPF1-treated mice comparedto vehicle-treated mice.

FIGS. 45A and 45B show that there was a significant increase in pastagnawing in the PPF1-treated group compared to the vehicle-treated group3 weeks after the last treatment, indicating motor improvement (FIG.45B). The test was developed to study motor deficits in small rodents.Animals were brought into the experimental room at least 2 hours priorto testing. The cage top, water bottle, and food pellets were removedand a small piece of dry spaghetti (approx. 5 mm) was placed in thecage. A microphone was placed above the noodle pieces. Recording wasinitiated as soon as an animal started to eat. The number of bites pergnawing episode and the biting frequency were evaluated, and the gnawingpattern analyzed using Avisoft SASLab Pro software. All data shown aremean±s.e.m. (*P<0.05 by unpaired t-test).

FIG. 46 shows the results of a wire suspension test, which assessesneuromuscular abnormalities of motor strength. There was a significantincrease in time to fall in the PPF1-treated group compared to thevehicle-treated group 3 weeks after the last treatment. To perform thetest, the wire cage lid was used and duct tape placed around theperimeter to prevent the mouse from walking off the ledge. The animalwas placed on the top of the cage lid. The lid was lightly shaken threetimes to force the mouse to grip the wires and then the lid turnedupside down. The lid was held at a height of approximately 50-60 cmabove a soft underlay, high enough to prevent the mouse from jumpingdown, but not high enough to cause harm in the event of a fall. Thelatency to fall down was quantified and a 300-second cut-off time used.Normally, a wild-type mouse can hang upside down for several minutes.

FIGS. 47A, 47B, and 47C depict the results from a beam walk test. FIG.47A shows the different beam shapes and sizes (square or cylindricalrods) used in five different trials of increasing difficulty. FIG. 47Bdepicts the results of the five trials 72 hours after the lasttreatment. FIG. 47C depicts the results of the five trials 3 weeks afterthe last treatment. Mice treated with PPF1 showed significantly highersuccess at traversing the beam during Trial 5 of Testing 1 (72-hourpost-treatment) and during Trial 4 of Testing 2 (3 weekspost-treatment). All data shown are mean±s.e.m. (**P<0.01 by binomialtest).

FIGS. 48A through 48F show histological results of striatal andhippocampal staining. FIG. 48A reports striatal CD68 staining. FIG. 48Breports hippocampal CD68 staining. FIG. 48C reports striatal Iba-1staining. FIG. 48D reports hippocampal Iba-1 staining.

FIG. 48E reports striatal NeuN staining. FIG. 48F reports hippocampalNeuN staining. These figures show that mice treated with PPF1demonstrated decreased microgliosis (neuroinflammation) by Iba-1 andCD68 in both the striatum and the hippocampus and increased neuronalsurvival by NeuN staining in the striatum and hippocampus. All datashown are mean±s.e.m. (*P<0.05, **p<0.01, ***P<0.001 by unpairedt-test).

u. Example 21

PPF1, HAS1, or saline control were administered to 12-month-old mice(NOD.Cg-Prkdscid Il2rgtm 1 Wj1/SzJ, “NSG” strain). HAS1 is acommercially-available HAS with over 95% human albumin (in relation tototal protein) in a 5% solution (w/v, 50 g/L), prepared by a coldalcohol fractionation method, and derived from pooled human plasma fromdonors. Except where noted, HAS1 was administered in the examples hereinin vivo using the 5%. Mice were injected by IV administration of withPPF1, HAS1, or sterile saline at 150 μL per dose daily for 7 consecutivedays and analyzed behaviorally 4 weeks after the last dose.

FIG. 49 reports Barnes Maze escape latency for a mouse to enter theescape hole for PPF1, HAS1, and vehicle-treated mice. PPF1 treatedanimals found the escape hole significantly faster than vehicle-treatedanimals. This data shows that PPF1 efficiently enhances cognition inaged NSG animals, while HAS1 treatment has no effect onhippocampal-dependent memory. All data shown are mean±s.e.m. (*P<0.05 byunpaired t-test).

v. Example 22

Clinical Paradigms Using PPF.

(1) Mild-to-Moderate AD.

Men and women 60 years or older with mild-to-moderate AD are randomlyallocated to receive 100 mL or 250 mL once daily of PPF1 for 5 days(“pulsed dosing”) during weeks 1 and 13 of the study with a totalduration of 6 months. During the two 5-day dosing periods, subjectsreside in inpatient observation units to facilitate safety evaluation,and all subjects undergo a screening visit, baseline visit, treatmentvisits, follow-up visits, and an end of study/early termination visit.Safety and tolerability assessments occur at every visit. Neurocognitiveand motor assessments are performed at baseline and at periodic interimvisits following dosing.

Primary endpoints are safety, tolerability, and feasibility of eachdosing regimen. Safety is measured by the incidence oftreatment-emergent adverse events. Tolerability is measured by thenumber of subjects completing 8 weeks after receiving at least 5infusions and subject completing 24 weeks after receiving at least 10infusions. Study feasibility is measured by the number of subjectscompleting 5 and 10 infusions. Secondary endpoints assess potentialeffects on cognition using various established cognitive measuresincluding the Alzheimer's Disease Assessment Scale-Cognitive Subscale.Exploratory endpoints include assessment of changes in composition anddistribution of blood-based biomarkers, as well as changes in magneticresonance imaging.

(2) Mild-to-Moderate AD.

Two groups of subjects diagnosed with mild to moderate AD are randomizedto active treatment in a double-blind manner. All subjects receive oneinfusion per day at the randomized dose for 5 consecutive days duringweeks 1 and 13 with a study duration totaling 6 months. Subjects arerandomized to one of the following two dose levels: 100 mL and 250 mL ofPPF1. Dosing groups are also stratified by gender. Administrationduration is 2-2.5 hours, and flow rates titrated according todose-specific guidelines so that the entire dose is administered.

Subjects participate in optional CSF biomarker research. Such subjectsundergo two lumbar punctures for CSF collection, the first prior toinitial dosing, and the second following final dosing. Neurocognitiveand motor assessments are performed at baseline and periodic interimassessments performed following dosing.

Safety, tolerability, and feasibility of each dosing regimen aredetermined. Cognitive scores are determined and summarized over thestudy, including: Mini-Mental State Examination (MMSE); 11-itemAlzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog/11);Grooved Pegboard Test; Category Fluency Test (CFT); Clinical DementiaRating Scale—Sum of Boxes (CDR-SOB); Alzheimer's Disease CooperativeStudy—Activities of Daily Living (ADCS-ADL); Alzheimer's DiseaseCooperative Study—Clinical Global Impression of Change (ADCS-CGIC);Neuropsychiatric Inventory Questionnaire (NPI-Q); and SavonixNeurocognitive Assessments and Digit Span.

(3) Parkinson's Disease.

Subjects with Parkinson's Disease and cognitive impairment arerandomized to two groups: 2 periods of active treatment and placebo.Subjects receive one infusion per day of active or placebo treatment for5 consecutive days (“pulsed dosing”) during the study's first week.During week 13, both groups receive active treatment for 5 consecutivedays, and the study duration is approximately 7 months. Administrationduration is 2-2.5 hours, and flow rates titrated according todose-specific guidelines so that the entire dose is administered.

Safety, tolerability, and feasibility of each dosing regimen aredetermined. Cognitive and motor function are summarized over the study,including: MoCA; Continuity and Power of Attention, Working Memory, andEpisodic Memory on the CDR-CCB; MDS-UPDRS3; MDS-UPDRS2; SE-ADL, andCISI-PD.

It is to be understood that this invention is not limited to particularaspects described, as such may vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularaspects only, and is not intended to be limiting, since the scope of thepresent invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual aspects described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalaspects without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and aspects of the invention as well as specificexamples thereof, are intended to encompass both structural andfunctional equivalents thereof.

Additionally, it is intended that such equivalents include bothcurrently known equivalents and equivalents developed in the future,i.e., any elements developed that perform the same function, regardlessof structure. The scope of the present invention, therefore, is notintended to be limited to the exemplary aspects shown and describedherein. Rather, the scope and spirit of present invention is embodied bythe appended claims.

The invention claimed is:
 1. A method of treating a subject diagnosedwith a cognitive impairment, the method comprising, administering aneffective amount of a Plasma Fraction using a Pulse Dosed dosingregimen, wherein the Pulse Dosed dosing regimen comprises at least 3 to14 consecutive days of administration.
 2. The method of claim 1 whereinthe Plasma Fraction is a Plasma Protein Fraction.
 3. The method of claim2 wherein the Plasma Protein Fraction comprises between 83% to 95%albumin.
 4. The method of claim 3 wherein the Plasma Protein Fraction isa commercially available Plasma Protein Fraction.
 5. The method of claim1 wherein the Plasma Fraction is a protein-enriched plasma proteinproduct.
 6. The method of claim 1 wherein the Plasma Fraction is derivedfrom plasma from a pool of young individuals.
 7. The method of claim 1wherein the Plasma Fraction is produced from a mammalian blood product.8. The method of claim 7 wherein the mammalian blood product is a humanblood product.
 9. The method of claim 7 further comprising monitoringthe subject for improved cognitive function.
 10. The method of claim 7wherein the subject is a mammal.
 11. The method of claim 10 wherein themammal is a human.
 12. The method of claim 7 wherein the Plasma Fractionis derived from a pool of young individuals.
 13. The method of claim 7wherein the Pulse Dosed dosing regimen comprises administering thePlasma Fraction for five to seven consecutive days.
 14. The method ofclaim 7 wherein the subject follows an exercise regimen after the PulsedDosed dosing regimen has been fully administered.
 15. The method ofclaim 1 further comprising monitoring the subject for improved cognitivefunction.
 16. The method of claim 1 wherein the subject is a mammal. 17.The method of claim 16 wherein the mammal is a human.
 18. The method ofclaim 1 wherein the Pulse Dosed dosing regimen comprises administeringthe Plasma Fraction for five to seven consecutive days.
 19. The methodof claim 1 wherein the subject follows an exercise regimen after thePulsed Dosed dosing regimen has been fully administered.